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In Vitro Plantlet Regeneration from the Bulbs of Shallot (Allium Cepa Var. Group Aggregatum)

Bantewalu Hailekidan, Mebeasilassie Andargie*, Kebebew Assefa

Biology Department, College of Natural and Computational Sciences (CNCS), Haramaya University, Dire Dawa, Ethiopia

Abstract: The study was undertaken to develop an efficient protocol for in vitro regeneration of shallot (Allium cepa). Two local shallot varieties (Huruta and Minjar) were used as experimental materials where basal discs were used as explants. Murashige and Skoog medium supplemented with different concentrations and combinations of 2, 4-Dichlorophenoxyacetic Acid, 6-Benzylaminopurine, Kinetin and α-naphthaleneacetic acid were used for callus induction and regeneration of plantlet. Maximum callus induction was observed in genotype Huruta (81.11%) in medium supplemented with 1mg/l 2, 4- Dichlorophenoxyacetic Acid. In combined effect both genotypes Huruta and Minjar showed highest callus induction (74.44%) from basal discs placed in medium supplemented with 1 mg/1 2,4- Dichlorophenoxyacetic Acid. Among the different types and combination of plant growth regulators, the maximum callus fresh weight of 1.26 and 1.20 g were achieved with 1 mg/l 2, 4- Dichlorophenoxyacetic Acid and α-naphthaleneacetic acid combined with 1 mg/l 6-Benzylaminopurine, respectively. Regenerated plants were obtained via somatic embryogenesis and organogenesis. Murashige and Skoog medium supplemented with 5.0 mg/l 6-Benzylaminopurine + 0.1 mg/l α-naphthaleneacetic acid showed higher percentage of shoot regeneration (91.11%). 1.5 mg/l indole-3-butyric acid + 2 mg/l 6-Benzylaminopurine was the optimum concentration giving 86.66% of rooted plantlets. The survival rate of transferred regenerated plantlets was satisfactory 66.6% and 60.0% for Minjar and Huruta respectively.

Keywords: callus, MS medium, plant regeneration, shallot, somatic embryogenesis

Received August 09, 2013; Revised October 20, 2013; Accepted November 14, 2013

Citation: Bantewalu Hailekidan, Mebeasilassie Andargie, Kebebew Assefa, In Vitro Plantlet Regeneration from the Bulbs of Shallot (Allium Cepa Var. Group Aggregatum). Research in Plant Sciences, 1 (2). 45-52. January 2013. doi:10.12691/plant-1-2-7

1. Introduction

Allium is the largest and important representative genus of the Alliaceae family and comprises 450 species, widely distributed in the northern hemisphere [1]. Shallot (Allium cepa var aggregatum Don. 2n=16) is an important vegetable crop belonging to the family Liliaceae. It is originated in tropical central or western Asia and has been cultivated from very early period [2]. The allium spp. are distributed widely through the temperate, warm temperate and boreal zone of the northern hemisphere. Typically alliums are plants of open sunny dry site in fairly arid climates [3]. As shallot and its relative species are generally open pollinated crops and have been cultivated for long times, a number of landraces and natural hybrids either intra or inter-specific are on the increase [4]. It is among the most important vegetable species in the world and an essential ingredient used in every home daily. It is hardly possible to get a dish without this vegetable in every meal of a day [5]. It serves human being both in the therapeutic as well as culinary purposes [6].

In vitro plant regeneration from cells, tissues and organ cultures is a prerequisite for the application of plant biotechnology to plant propagation, plant breeding and genetic improvement. It is the only technology for the production of large quantities of ‘‘elite’’ planting material so as to increase the production and productivity. Micropropagation is advantageous over traditional propagation as it can be used to multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds or respond well to vegetative reproduction. It also leads to simultaneous accomplishment of rapid large-scale propagation of new genotypes [7].

The conventional method of clonal propagation of shallot is by bulbs. This method, aside from being slow and seasonal, has resulted in a geometric increase in the transmission of diseases and uncontrollable spread of diseases, more importantly viruses, which cause as high as 45% reduction in yield. So an alternative method to conventional asexual propagation of shallot is by tissue culture or micropropagation since it is convenient for propagating novel genotypes.

The establishment of efficient and promising protocol for in vitro plantlet regeneration of shallot is required for the application of modern biotechnological tools, such as asexual reproduction of elite stocks, recovery of useful somaclonal variants, germplasm preservation as well as the production of transgenic plants with improved agronomic traits, and interspecific hybrids [8,9]. Tissue culture has also been effective in most species of Alliaceae including Allium sativum, Allium ampeloprasum, Allium fistulosum, Allium hirtifolium, Allium tuberosum, Allium chinense and others [10,11,12,13]. There have been previous works on onion in vitro culture with regard to callus production and shoot regeneration from twin scales [14,15]; however, there is a real need for clones of economically important Allium crops like garlic and shallots, which generally do not propagate by seeds, as well as leek and onion for breeding programs [14]. Few attempts have been made on in vitro regeneration in the genus Allium, and most of the reports are focused on Allium cepa var. ascalonicum [16,17,18,19] and Allium scorodoprasum spp rotundum [20]. An established micropropagation protocol is not only important for the maintenance and mass production of clean stocks of commercially important cultivars but also in future research work in shallot/onion breeding which include field testing and selection, disease elimination, germplasm collection, conservation and evaluation. In this study, we present a protocol for in vitro regeneration of Allium cepa Var. group aggregatum from bulbs resulting in higher number of shoots per cultured explants.

2. Materials and Methods

2.1. Preparation of Explants

Two varieties of A. cepa, namely Huruta (DZSHT-91) and Minjar (DZSHT-164-1B) were obtained from Debre-Zeit Agricultural Research center, Debre-Zeit, Ethiopia. Fresh and undamaged bulbs were washed in tap water and liquid soap in order to clean the soils and some contaminants from the bulbs, then treated with sterile water containing two drops of Tween 20 per 100ml and 20% bleach for 15-20 minutes with contunuous agitation and finally rinsed 5-10 times with distilled water. In order to remove the fungicides and other residues, the explants were washed in tap water, and then washed in distilled water three times. Then the outer dry scales were peeled off and rejected. Lower halves of bulbs containing the basal disc were washed thoroughly and immersed in 70% alcohol for 1-2 min, rinsed four times in distilled water, surface sterilized in 5.25% sodium hypochlorite (NaOCl) solution for 15 min and again rinsed four times in autoclaved double distilled water. The explants which were excised from the bulbs were inoculated in MS medium [21] containing 3% (w/v) sucrose and 0.8% (w/v) agar, pH was adjusted to 5.7. The callus induction medium was supplemented with 1mg/l 2, 4-D, NAA, Kn and BAP. All media were autoclaved at 121°C, 1.05kg cm⁻² pressure for 15 min. Cultures were incubated at room temperature of 25±2°C and under Phillips fluorescent day light tubes emitting 3200 Lux for 16/8 h light/dark period.

2.2. Shoot Multiplication

For multiplication, after the calli attain a convenient size, sub culture were done in the MS [21] media containing different combinations and concentrations of either BAP (1.0, 3.0, and 5.0 mg/l) or Kn (0.1, 0.5 and 1.0mg/l) alone as well as 0.1 mg/l NAA in combination with BAP (1.0, 3.0 and 5.0 mg/l) [22]. Cultured petridishes were incubated as explained above.

2.3. In vitro Rooting and Acclimatization

The elongated shoots (2-3 cm) were rescued aseptically and transferred to root induction medium containing various concentrations of indole-3-butyric acid (0.5, 1.0 and 1.5mg/l) and in combination with 2mg/l of BAP. The vials containing plantlet were incubated under continuous light. Day to day observation was carried out to note the response of the growing plantlets. The well-rooted shoots were removed and washed thoroughly with distilled water. The plantlets were transplanted in perforated plastic cups filled with potting mixture of soil, sand and FYM (Farm yard Manure) (1:2:1) and irrigated with water daily in a green house. Finally, the hardened plants were transferred to earthen pots.

2.4. Statistical Analysis

All experiments were setup in the completely randomized design with three replicates, each treatment consisted of 50 explants and data were analyzed using one-way analysis of variance (ANOVA) at the 0.05 significance level (SAS Institute, Cary, NC, USA).

3. Results and Discussion

Despite the economic importance of shallots, work on development of plant regeneration systems for Allium cepa var aggregatum has not progressed as in the case of several other Liliaceae crops.

3.1. Callus Induction

In the present study, callus induction potential of the two shallot varieties was tested in different hormone and hormonal combination. Callus initiation started from 28.7 to 30.2 days of incubation in Huruta and Minjar, respectively for over the hormonal treatments. Despite the length of day to induce callus Minjar induce more callus and also had high mean fresh weight than Huruta (Table 1).

From the present investigation it was evident that the MS medium which consist 2,4-D showed the highest callus induction (74.44%) which required minimum (28.47) days for callus initiation (Table 2), followed by NAA + BAP , 2,4-D + Kn and BAP whereas NAA and Kn alone showed the lowest callusing. The highest fresh callus weight is found in a media that contain 2, 4-D (Fig. 1). Enhancing capacity of 2, 4-D on onion (Allium cepa L.) and garlic (Allium sativum L.) at different concentration was reported earlier in the callus formation and subsequently in the in vitro regeneration [23,24,25,26,27]. A wide range of variation was also found in callus induction ability and fresh weight among the interaction of varieties to hormones (Table 1). The highest fresh weight was found in 2,4-D*Minjar followed by 2,4-D*Huruta and the least was for NAA* Minjar. 81.11% of the explant responds callus in Huruta*2, 4-D followed by 76.66% for Minjar*NAA + BAP. In both cases, the hormone free media did not show any response.

Table 1. Mean interaction effect of different types and combinations of plant growth regulators on callus induction of the two shallot varieties.

Table 2. Effect of plant growth regulators on callus induction of cultured A. cepa explants

3.2. Shoot Regeneration from Callus

It is evident from the experiment that percent of shoot regeneration and number of shoots per callus increased with the increasing concentration of BAP up to 5mg/l (Table 3). The highest shoot regeneration was found in media containing 5.0 +0.1NAA (91.11%) (Table 4 and Figure 2). There was also a great variation in the number of shoot per callus and shoot height between the hormonal treatments for both the varieties. The highest number of shoot per callus is found on the media containing no hormone (6.85) but all the shoots were albino shoots (Figure 3) whereas the lowest no of shoot was found in a media containing 1.0mg/l BAP+0.1mg/lNAA and 1.0mg/l BAP. In terms of shoot height the albinos which are found in a media where there was no hormone had the lowest height as compared to 3.0mg/l BAP+0.1mg/lNAA and 5.0mg/l BAP. Earlier report by [28] showed an increase in percent shoot formation and number of shoot per explant was observed when the concentration of BAP+NAA was increased and in the current study it is also confirmed that cytokinins were the most determining hormones involved in the regeneration of shoots in shallot. Explants cultured with BAP produced significantly more shoots than those cultured without BAP. The addition of BAP has increased the proliferation rate of a member of Allium spp. [29,30,31,32,33,34].

Table 3. Mean interaction effect of different types and combinations of growth regulators on shoot regeneration from callus

Table 4. Effect of plant growth regulators on shoot regeneration of cultured A. cepa explants

Figure 1. Calluses induced from diffrent hormones for the two varieties; A- Huruta with 2,4- D+Kn, B and C- Minjar with 2,4-D, D-Minjar with Kn, E- Huruta with 2,4-D, F-Huruta with 2,4-D+BAP

Figure 2. shoot regenerated from different hormone concentration for the two varieties: A, B, C Huruta with shooting media containing diffrent concentration of BAP; D, E, F Minjar with shooting media containing diffrent concentration of BAP; G, H Huruta with shooting media containing diffrent concentration of BAP+NAA and I, J Minjar with shooting media containing diffrent concentration of BAP+NAA.

Figure 3. Albino shoots regenerated from calli of shallots cultured on hormone-free MS media

In this study the increase in Kinetin from 0.1-1.0mg/l decreases the percent shoot induction from 87% to 67% while [35] reported that as the concentration of kinetin increases from 1-10 mg/l, the frequency of the plantlet regeneration increases from 43% to 55%. Differences in the requirement for growth regulators might be due to the differences in the type of explants and in the cultivar used. In another report by [36] percentage of shoot formation was also higher (80%) in shoot tip explants cultured in MS media supplemented with 1.5 mg L⁻¹ kinetin; however, the shoot formation percentage was decreased with the increase in concentration of kinetin which is in agreement with our finding. [37] also reported that maximum rate of shoot regeneration in Lavender species was observed when MS medium was supplemented with 1mg/L Kinetin. The significant decline observed in shoot induction with increase in the hormonal concentration indicating its inhibitory response on shoot regeneration.

According to [38] when kinetin 2.0 was used as shoot regeneration medium, shoot formation (5-8 shoots per piece of callus) was obtained whereas BAP used at higher concentrations, lead to albino shoot formation in some calli. But in the current study even if there were albino shoot formations, it did not come from the higher concentration of BAP rather they were observed in a media where there was no hormone. Chlorophyll deficiency or albinism is a standard marker in plant cytoplasmic genetics. Its stability is consistent with mutations in the plastid genome because nuclear mutation induces plastid ribosome deficiency [39]. The frequency of albino plant regeneration depends on several factors such as genotype [40], explant age [41] and medium composition [42].

Multiple shoot regeneration was observed from 28.44 to 38.83 days of culture in the current study which is in agreement with an earlier report by [43] in Allium ampeloprasum which took five to six weeks. It was worth noting that plant regeneration depended on several factors including the genotypes, hormone supplements and so forth. Statistical analysis revealed that significant variation was observed among varieties, hormonal concentrations and their interaction for days of induction, percent shoot induction and number of shoot per explant (Table 3).

3.3. Rooting of A. cepa Shoots and Acclimatization

Well root induction is very important for establishment of in vitro regenerated plantlets. Induction of shoots or roots was dependent on the cytokinin/auxin combination [44]. From the present study, it was evident that the highest root initiation was observed in 1.5mg/l IBA+2mg/l BAP (86.66%) and the lowest was found in 0.5 IBA+2BAP in addition the hormone free media didn`t induce any root (Table 5 and Figure 4). A maximum mean number of roots 4.91 and 4.63 were counted for media containing 1.5mg/l IBA and, 1.0mg/l IBA+2mg/l BAP respectively. The least mean number of roots were observed in 0.5mg/l IBA. Concerning the length of the roots, media containing 1.5mg/l IBA+2mg/l BAP and 1.5mg/l IBA induced a maximum mean length of 1.79 and 1.71 respectively and media containing 0.5mg/l IBA produced minimum mean length of root (1.38) for hormonal treatments over the two varieties.

Table 5. Effect of plant growth regulators on root regeneration of cultured A. cepa explants

Figure 4. A, B, Huruta with rooting media containing 1.0mg/l IBA and 1.5mg/l IBA;C, D Minjar with rooting media containing 1.0mg/l IBA and 1.5mg/l IBA

According to the mean of the two varieties for over all hormonal treatments, Minjar leads by early inducing and by inducing more root while Huruta gives large number of roots per explant and having larger root length than Minjar. Here it is also noticed that no root proliferation was observed in medium without any growth regulators. Therefore, it is proved that the use of phytohormone is essential for root multiplication and prolfieration. The findings are in agreement with those observed in other Allium species [45,46,47]. Effectiveness of IBA + BAP for in vitro root regeneration from different plant part cultures was also reported in several other plants [48,49,50,51]. In addition, being of a stable nature, IBA is the preferred auxin for adventitious root initiation in many plant species [52,53,54,55] Uranbey, 2010a; Uranbey, 2010b).

After satisfactory growth of root system, the plantlets were removed from vessels and transferred into soil in small pots and the pots were transferred into greenhouse for proper hardening. Survival rate of the acclimatized plantlets was 66.6% and 60.0% for Minjar and Huruta respectively (Figure 5).

Figure 5. Acclimatized plantlet of the two shallot varieties: A (above) aclimatized plantlets of the variety Huruta; and B (below) aclimatized plantlets of the variety Minjar

3.4. Conclusion

In the present study, it was observed that 2, 4-D was found as the best auxin for callus induction in Allium cepa Var. group aggregatum. The best conditions for in vitro shoot and root induction using bulb explants of A. cepa was the treatment with 5.0 + 0.1NAA and 1.5mg/l IBA+ 2mg/l. It is concluded that the manipulation of culture conditions using various combinations and concentrations of growth hormones can provide a reproducible protocol and reduce the high costs of hybrid seed production.

Acknowledgement

The authors are grateful to the Ministry of Education of Ethiopia (MOE) for the financial support to carry out this work.

Statement of Competing Interests

Authors have declared that no competing interests exist.

List of Abbreviations

BAP 6-Benzylaminopurine

2, 4-D2, 4-Dichlorophenoxyacetic Acid

IBAindole-3-butyric acid

KnKinetin

MSMurashige and Skoog

NAAα-naphthaleneacetic acid

PGRsPlant growth regulators

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