British Biotechnology Journal 8(3): 1-12, 2015, Article no.BBJ.18619 ISSN: 2231–2927

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In vitro Plantlet Regeneration of Four Local ( sativum) Accessions of Bangladesh

Sayed Raihanul Haider1, Mohammad Rashed Hossain1, Shanjida Rahman1, Shirin Sultana1, Tamanna Quddus1, Moutoshi Chakraborti1, Aunamika Hoque1, Md Hasan Shahriar1 and Md Ashraful Haque1*

1Department of Genetics and Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Authors’ contributions

This work was carried out in collaboration between all authors. Authors SRH and MRH contributed equally to this work. Authors SRH, MRH, SR and MAH designed the experiment. Authors SRH conducted the lab work and authors SR, SS, TQ, MC, AH and MHS helped in conducting the lab work, collecting the data and interpreting the results. Authors SRH and MAH analysed the data. Authors SR and MHS prepared the initial manuscript which was extensively edited by authors MRH and MAH. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/BBJ/2015/18619 Editor(s): (1) Mahalingam Govindaraj, ICRISAT, Patancheru, India. Reviewers: (1) B. V. Tembhurne, Department of Genetics and Plant Breeding, University of Agricultural Sciences, Raichur, Karnataka, India. (2) Danielle Camargo Scotton, Laboratory of Plant breeding, University of São Paulo, Brazil. (3) Anonymous, Lille University of Science and Technology, France. Complete Peer review History: http://sciencedomain.org/review-history/10000

Received 1st May 2015 th Original Research Article Accepted 27 May 2015 Published 30th June 2015

ABSTRACT

Aims: The genetic improvement of garlic can be achieved by biotechnological manipulations as breeding in this vegetatively propagated crop is limited. The current research was conducted with a view to develop an efficient in vitro regeneration protocol for four local garlic accessions namely, G121, G122, G123 and G124. Place, Duration and Design of Study: The experiment was conducted in the Tissue Culture Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University during the period from June 2013 to June 2014 using three-factorial experimental design. Methodology: The root tips, basal disc and leaf base were cultured in MS medium supplemented with 2, 4-Dichlorophenoxyacetic acid (2, 4-D) alone, and with both 2, 4-D and 6-Benzylaminopurine

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*Corresponding author: Email: [email protected];

Haider et al.; BBJ, 8(3): 1-12, 2015; Article no.BBJ.18619

(BAP) together for callus induction and the later for subsequent sub-culturing and proliferation of callus. MS medium supplemented with 1-Naphthaleneacetic acid (NAA) and BAP was used for plantlet regeneration. Results: The percentage of callus induction increased with the increase in the concentration of 2,4- D, starting from 0.5 mg L-1 till 2.0 mg L-1 and declined with further increase in the concentration of 2,4-D. The MS medium supplemented with 2,4-D and BAP showed higher percentage of callus induction and callus proliferation compared to that of with 2,4-D alone. The highest percentage of callus induction was observed in the genotype G124 from the explant basal disc (85%) and in the genotype G121 from the explant leaf base (80%) with 2.0 mg L-1 2,4-D and 2.0 mg L-1 BAP. MS medium supplemented with 2 mg L-1 2,4-D + 0.5 mg L-1 BAP showed highest percentage of callus proliferation (90%) in almost all the genotypes. The highest percentage of plantlet regeneration were observed in the genotype G124 for the explants basal disc (63.33%) in MS medium supplemented with 2 mg L-1 NAA + 1 mg L-1 BAP. The survival rate of the plantlets after acclimatization varied from 40% (in G123) to 70% (in G121). Conclusion: The optimized protocol of plant regeneration from local garlic accessions will be useful for any future garlic improvement programs using biotechnological means.

Keywords: Garlic; tissue culture; callus induction; growth medium & phytohormone.

1. INTRODUCTION the problem of low propagation rate and continuous accumulation of different deleterious Allium sativum, commonly known as Garlic is an viruses [19-26]. Virus free clones producing aromatic bulbous plant and an herbaceous through meristem culture showed higher yield annual spice belonging to the subfamily with improved quality [27]. Therefore, the tissue under family . It is culture techniques have got high potential for the cultivated for its both culinary and medicinal uses improvement of garlic in respect of yield and [1-3]. It has biological applications as like quality [28]. As its sexual reproduction in Garlic is antibiotic, anticancer, antithrombotic and in lipid restricted and is associated with some genetic lowering cardiovascular disorders [4-12]. It is complications, the tissue culture can be used for diabetes [13] and sickle cell anemia [14] employed for its propagation and genetic patients. In Bangladesh, 165 thousand metric ton improvement research [29]. This research of garlic is produced with an per acre yield of program has thus been undertaken to establish 1795 kg every year. Among the Asian countries, an efficient in vitro regeneration protocol for Bangladesh has lowest yield of garlic [15] and garlic from root tip, leaf base and basal disc as the yield is gradually decreasing probably due to explants using four locally grown garlic prevalence of viral infection [16]. The bulb yield accessions. We also aimed at exploring the was reduced by 20 to 60% due to virus infection variability of in vitro responses among different and by 80% due to mixed infection depending on garlic accessions in various combinations and cultivar and stage of infection [17]. The concentration of growth regulators. regeneration of from tissue culture is imperative and essential technique techniques of 2. MATERIALS AND METHODS biotechnological research and sometimes genetic manipulation of plants are achieved The experiment was conducted in the Tissue through this technique. Garlic is mainly Culture Laboratory of the Department of propagated through vegetative means and the Genetics and Plant Breeding, Bangladesh improvement of garlic through breeding Agricultural University, Mymensingh. Healthy programs is limited due to difficulties of inducing growing root tips, basal disc and leaf base of the flowering [18]. The application of biotechnology sprouted cloves of four local garlic accessions in combination with the conventional breeding namely, G 121, G122, G 123 and G 124 were method will thus be useful for any garlic used as explants. The explants were sterilized improvement research programs for which an initially with 70% ethanol for 1 minute followed by optimized protocol of in vitro regeneration is a HgCl2 for 3 minutes. The pH of the MS medium pre-requisite. Over the last decades, efforts have [30] was adjusted to 5.8±0.02 with 1 N HCl or 1 been made to develop in vitro techniques for N NaOH solutions and then autoclaved at 121ºC garlic micro-propagation via somatic and 15 psi for 15-20 min. MS medium embryogenesis and organogenesis to mitigate supplemented with 0.5, 1.0, 1.5, 2.0, 2.5 mg L-1

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2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5, concentrations of 2,4-D [34]. Effect of five 1.0, 1.5, 2.0, 2.5 mg L-1 2,4-D + 2.0 mg L-1 6- different concentrations of 2,4-D viz., 0.5, 1.0, Benzylaminopurine (BAP) was used for induction 1.5, 2.0 and 2.5 mg L-1 on callus induction was of callus and the later for subculturing and studied and significant variation was observed proliferation of callus. Inoculated explants were between the genotypes, hormones, explants and incubated both in dark and light condition under all their interactions except the interaction controlled temperature (25±2ºC) and a between explants and hormone concentrations photoperiod of about 16 h with a light intensity of (Table 1). Percent callus induction was increased 2000 – 3000 lux. The calli of optimum size was in all four genotypes with the increase in the then cultured for plantlet regeneration in MS concentration of 2,4-D starting from 0.5 mg L-1 to medium supplemented with 0.5, 1.0, 1.5, 2.0 mg 2.0 mg L-1 for all three explants (Fig. 1). L-1 1-naphthaleneacetic acid (NAA) + 1.0 mg L-1 However, the callus induction ability decreased BAP. The regenerated plantlets were then placed with a further increase in the concentration of into 10 cm plastic pots containing ground soil and 2,4-D (i.e., at 2.5 mg L-1 2,4-D) as a clear rotten cowdung in a ratio of 1:1 for acclimatizing declining trend was observed in all the genotypes the plantlets in vivo condition in growth room for for all three explants. Similar trends in percent 7 days. When the plantlets became 4-8 cm callus induction were also reported previously height with 3-6 well developed leaves and roots, with varying reports of the concentration of 2,4-D the plantlets were transplanted into pots that induced the highest percentage of callus containing the above mentioned potting mixture [12,15,29]. For example, our results suggests which was then transferred to the field when the that the highest percentage of callus (75%) is plantlets appeared to be self-sustainable. The induced in the genotype G121 for both the experiment was set following three-factorial explants, leaf base and basal disc at 2.0 mg L-1 experimental design. Data on percent callus of 2,4-D whereas the concentration 1.5 mg L-1 induction, proliferation and regeneration were was shown to induce higher percentage of callus recorded using standard protocol [31-33]. The in two varieties namely local and exotic [12]. The callus proliferation was measured in terms of callus induction percentage of the explant root tip percent of the increase in fresh weight after 20 was lowest compared to the other explants days of subculture [33]. The data were analyzed (Fig. 1). However, the root tip of the genotype G (percentage, Analysis of variance) using Minitab 124 showed highest percentage of callus software version 15.2 and the Least Significant induction compared to other genotypes, even Difference (LSD) was used for pair-wise though the performance of this genotype for comparison of means. other explants was poor (Fig. 1). The images of callus initiation are shown in Fig. 2. 3. RESULTS AND DISCUSSION The efficacy of the MS medium for indirect 3.1 Variability between Genotypes, organogenesis via callus induction, proliferation and regeneration in Allium is well established for Explants, Phytohormones and Their a range of explants such as seed, embryo, root Interactions tip, basal disc, shoot, young bulb and inflorescence etc. [35-39]. However, the other The leaf base, root tip and basal discs of four medium such as BDS, B5 and N6 has also been local accessions namely, G 121, G 122, G123 reported to show potentiality in indirect and G 124 were tested for their callus induction organogenesis of garlic and other genus of and plantlet regeneration ability under different species Allium [19,21,40-42]. Nagasawa and concentrations and combinations of hormones. Finer (1988) first successfully demonstrated the Significant differential response was observed use of the plant growth regulator 2,4- between the genotypes, explants, hormonal dichlorophenoxyacetic acid (2,4-D) alone as the combinations and most of possible interactions source of auxin in the media for organogenic calli between these sources of variations for callus induction in Allium [43]. The hormone 2,4-D was induction and plantlet regeneration as revealed also successfully used for callus induction and by Analysis of variance (ANOVA). The detailed proliferation in other genus of Allium species results are shown in Table 1. [19,34,36,44-46].

3.2 Effect of 2, 4-D on Callus Induction Addition of cytokinin at a lower level along with the higher concentration of 2,4-D has shown The callus induction and propagation is enhancement in callus induction than 2,4-D suggested to be largely determined by the alone that prompted the use of combined auxin-

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cytokinin in callus culture [39]. Along with 2,4-D Prompted by these findings, we had also as auxin source, kinetin [37], picloram [47], as investigated the combined effect of auxin and the source of cytokinin has shown enhanced cytokinin in callus induction and proliferation in callusing in Allium species. However, the addition local garlic genotypes and the results are shown of BA [31,41,44,48]; or BAP [42] as the in the next section. alternative source of cytokinin along with 2,4-D proved best for two Allium species namely, A. ampeloprasum and A. cepa.

Fig. 1. Effect of 2,4-D on percent callus induction from three explants, namely leaf base (A), root tip (B) and basal disc (C) in four local garlic genotypes. Data recorded at 14-21 days after inoculation and presented as mean over three replicates

Fig. 2. Figures showing- (A1-4) the initiation of callus from root tips in MS medium supplemented with 2.0 mg L-1 2,4-D at 21 days after inoculation; (B1-4) the proliferation of callus derived from the leaf base in MS medium supplemented with 2.0 mg L-1 2,4-D and 0.5 mg L-1 BAP at 45 days after inoculation; (C1-4) the regeneration of multiple shoots from the proliferated calli derived from root tips in MS medium supplemented with 2.0 mg L-1 NAA and 1.0 mg L-1 BAP of four garlic accessions namely, G 121, G122, G 123 and G 124

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Table 1. Mean squares, F-tests (ANOVA) and coefficient of variation (CV) of genotypes, explants, hormones and their interactions for different in vitro regeneration parameters

Sources of Percent callus Percent callus induction Percent plant variation induction under under different regeneration under different concentrations of 2,4-D + different concentrations concentrations 2 mg L-1 BAP of NAA + 1 mg L-1 BAP of 2,4-D df Mean CV df Mean CV (%) df Mean CV (%) squares (%) squares squares Genotype (G) 3 6.62** 9.58 3 17.88** 7.58 3 30.45** 10.92 Explant (E) 2 65.84** 22.89 2 16.65** 6.94 2 20.88** 19.42 Hormone (H) 4 463.05** 11.96 4 437.01** 7.63 3 200.30** 12.29 G x E 6 4.57** - 6 1.40 NS - 6 1.35** - G x H 12 3.25** - 12 1.27 NS - 9 7.95** - E x H 8 8.11NS - 8 3.04** - 6 0.98NS - G x E x H 24 4.57** - 24 2.24** - 18 0.53NS - **= significant at 5% level of probability, NS = not significant, df = degrees of freedom; CV = coefficient of variation, ‘x’ indicates interaction

3.3 Effect of 2,4-D and BAP on Callus The combination of 2,4-D and BAP seemed to Induction enhance the percentage of callus induction in almost all the genotypes (Fig. 3) compared to that of with the hormone 2,4-D alone (Fig. 1). For Five different concentrations of 2,4-D viz., 0.5, example, the highest percentage of callus -1 1.0, 1.5, 2.0 and 2.5 mg L in combination with a induction was 85% in MS medium supplemented -1 fixed concentration of BAP (2.0 mg L ) were with combined 2,4-D and BAP whereas the value used in MS medium to observe the callus was 75% when 2,4-D alone was used. This induction frequency of four local garlic genotypes findings are thus in agreement with the notion using leaf base, root tip and basal disc as described in previous reports [39,42,50]. The explants and the details results are shown in addition of BAP also increased the callus Fig. 3. In this case also, the callus induction induction ability of the root tip explants. For increased with the increase in the concentration example, without BAP, the highest percentage of -1 -1 of 2,4-D, starting from 0.5 mg L till 2.0 mg L callus induction from root tip was 65% (in G 124) and declined with further increase in the whereas the addition of BAP increased it to 75% concentration of 2,4-D. The highest percentage (in the accessions G124 & G121) as shown in of callus induction was observed in the genotype Figs. 1 and 3. G 121 from the explant leaf base (80%), in the genotype G 124 from the explant basal disc Regarding the explants, the basal discs and leaf (85%) and in the both genotypes from the explants performed better in terms of percentage explant root tip (75%) under 2.0 mg L-1 2,4-D and of callus induction compared to that of root tip in 2.0 mg L-1 BAP. Mehta et al. [49] observed both media combinations; MS + 2,4-D and MS + higher shoot regeneration from the leaf petiole 2,4-D + BAP. Luciani et al. [21] has also found explants of garlic using MS medium that the basal plates along with meristems were supplemented with 0.25 mg L-1 2,4-D+ 0.5 mg L-1 highly responsive explants compared to BAP. Nasim et al. [32], also observed the immature umbels and root tips, as indicated by initiation of globular-stage embryo differentiation percentage of induced callus, growing callus and and maximum proliferation of embryos in various regenerating callus. Kizil et al. [51], however, has BAP and 2,4-D concentrations. Khan et al. [12], found the root tip explants as most suitable for observed higher percentage of callus induction in bulblet regeneration. Unlike the media MS medium supplemented with 1.5 mg L-1 2,4-D composition of our experiments, they have used and 5 mg L-1 Kinetin (Kn), an alternative of BAP MS medium containing 5.0 mg L-1 BAP plus 0.5 as cytokinin source. Luciani et al. [21], also mg L-1 NAA. Root tips were also found to be observed best response in terms of callus commonly used for direct or indirect plant induction (41%) in 0.045 µM 2,4-D and 4.43 µM regeneration from garlic [19,48,52-53]. These BAP, however, they have used Basal Dunstan indicates that the explant types are one of the and Short (BDS) medium instead of MS medium. most important factors affecting plant

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regeneration besides other factors such as BAP such as Roksana et al. [57] observed genotype, concentrations and combinations of highest mean shoot proliferation in treatment of growth regulators etc. [50]. 0.5 mg L-1 2ip (purine) and 0.25 mg L-1 NAA. In this study, the highest percentage of proliferation 3.4 Effect of 2,4-D and BAP on Callus (90%) was observed in genotype G-121 for all Proliferation three explants, in genotype G-122 for root tip as explants, in genotype G-123 for basal disc as In case of callus proliferation, different explants and in genotype G-124 for root tip and concentrations of 2,4-D viz. 0.5, 1.0, 1.5, 2.0 and basal disc as explants (Table 2). The -1 2.5 mg L along with a fixed concentration of combination of 2,4-D and BAP was also -1 BAP (0.5 mg L ) were added in MS media for all successfully used for callus proliferation in other genotypes using leaf base, root tip and basal genus of Allium species [31-32,58]. disc as explants and results are shown in Table 2. Percent callus proliferation was 3.5 Effect of NAA and BAP on Plant increased in all four genotypes with the increase Regeneration in the concentration of 2,4-D for all three explants. However, the callus proliferation ability One of the objectives of this study was to identify decreased with a further increase in the -1 the best explant as a part of efficient protocol concentration of 2,4-D (i.e., at 2.5 mg L 2,4-D) development of garlic regeneration. So sensitivity for the genotypes for all three explants. of selected explants to shoot and root Comparatively higher concentration of auxin (2.0 regeneration was observed. Induction of shoots mg L-1 2,4-D) along with single concentration (0.5 -1 and roots from unorganized calli of each of the mg L ) of cytokinin was found suitable for the explant for all the four accessions exploited MS proliferation of callus than that of lower medium supplemented with different concentration of auxin. This finding is in concentrations of auxin and cytokinin. Several agreement of with that of previous reports combinations such as 0.5 mg L-1 NAA + 1.0 mg [29,54-56]. L-1 BAP, 1.0 mg L-1 NAA + 1.0 mg L-1 BAP, 1.5

mg L-1 NAA + 1.0 mg L-1 BAP and 2.0 mg L-1 In current study, MS media supplemented with -1 2.0 mg L-1 of 2,4-D and 0.5 mg L-1 of BAP was NAA + 1.0 mg L BAP were used for this found as best combination for callus proliferation. purposes (Fig. 4). All the accessions showed highest performance of plant regeneration at MS Nasim et al. [32] observed highest percentage of -1 -1 proliferation after 8 weeks of culture on medium + 2.0 mg L NAA + 1.0 mg L BAP. The lowest -1 frequency of plant regeneration was recorded at containing a combination of 1.0 mg L BAP and -1 -1 the combination of MS + 0.5 mg L NAA + 1.0 0.25 mg L 2,4-D. Best rate of proliferation was -1 observed by Mehta et al. [49] in MS medium mg L BAP. The other combinations showed supplemented with 0.25 mg L-1 2,4-D and 0.5 mg moderate performance in plant regeneration (Fig. L-1 BAP. However, highest proliferation 4). These findings are in agreement with that of frequency was also recorded by many scientists by Haque et al. [59]. using different hormone other than 2,4-D and

Fig. 3. Effect of 2,4-D and BAP on percent callus induction from three explants, namely leaf base (A), root tip (B) and basal disc (C) in four local garlic genotypes. Data recorded at 14-21 days after inoculation and presented as mean over three replicates

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Table 2. Effect of 2,4-D and BAP on proliferation of callus derived from three explants of four garlic genotypes. Mean of callus proliferation percentage over three replications are represented. Data recorded at 30-45 days after inoculation.

MS medium G 121 G 122 G 123 G 124 supplemented with Leaf Root tip Basal Leaf Root Basal Leaf Root Basal Leaf Root Basal 2,4-D + BAP base disc base tip disc base tip disc base tip disc (mg L-1) + (mg L-1) 0.5 + 0.5 40 35 40 35 35 40 30 30 35 40 40 45 1.0 + 0.5 65 65 80 65 60 70 60 60 70 65 70 75 1.5 + 0.5 80 75 85 75 85 75 70 80 80 80 80 85 2 + 0.5 90 90 90 75 90 85 80 80 90 85 90 90 2.5 + 0.5 85 85 85 80 80 80 75 75 80 80 80 80 LSD (treatment) = 10.2; LSD (explant) = 1.69; LSD (genotype) = 4.535

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3.6 Survival Rate of the Regenerated hardening. Then plantlets were transplanted to Plantlets onto Soil earthen pots (Fig. 5). The survival rate of the plantlets varied from 70% (in G 121) to 40% (in G Regenerated plantlets were transplanted in small 123) in the pot mixture prepared with 50% loamy plastic pots into acclimatization room for proper soil and 50% cowdung (Table 3).

Fig. 4. Effect of different combination of NAA and BAP in MS medium on percent plant regeneration of four garlic genotypes. Data presented as mean over three replicates

Fig. 5. Figures showing- (A1-4) the regeneration of plantlets in MS medium supplemented with 2.0 mg L-1 NAA and 1.0 mg L-1 BAP; (B1-4) the acclimatization of plantlets in the hardening room and establihment of plantlets in pot after hardening of four garlic accessions namely, G 121, G122, G 123 and G 124

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Table 3. Survival rate of the regenerated plantlets onto soil

Garlic accessions No. of plantlets No. of plantlets Survival rate transplanted survived (%) G 121 10 7 70 G 122 10 5 50 G123 10 4 40 G 124 10 6 60

4. CONCLUSION 6. Borek C. Antioxidant health effects of aged garlic extract. The Journal of nutrition. The regeneration protocol for different explants 2001;131(3):1010-1015. of four local garlic accessions was optimized. It 7. Harris JC, Cottrell S, Plummer S, Lloyd D. was observed that the accession G 121 Antimicrobial properties of Allium sativum performed better in terms of callus induction, (garlic). Applied Microbiology and proliferation and plantlet regeneration followed by Biotechnology. 2001;57(3):282-286. the accession G 124. The MS medium 8. Rivlin RS. Historical perspective on the use supplemented with 2.0 mg L-1 2,4-D and 0.5 mg of garlic. The Journal of Nutrition. 2001; L-1 BAP showed highest percentage of callus 131(3):951-954. induction and proliferation, respectively and the 9. Lawson LD. Garlic: A review of its explants basal disc was found to be more medicinal effects and indicated active responsive for callus induction. The protocol thus compounds. Blood. 1998;179:62. optimized for the plant regeneration from local 10. Yang CS, Chhabra SK, Hong JY, Smith garlic accessions provides a successful and TJ. Mechanisms of inhibition of chemical reliable propagation technique of garlic in vitro toxicity and carcinogenesis by diallyl regeneration and will be of great use for any sulfide (DAS) and related compounds from garlic improvement programs by biotechnological garlic. The Journal of Nutrition. 2001; manipulations, in future. 131(3):1041-1045. 11. Banerjee SK, Maulik SK. Effect of garlic on COMPETING INTERESTS cardiovascular disorders: A review. The Journal of Nutrition. 2002;1(1):4. Authors have declared that no competing 12. Khan N, Alam MS, Nath UK. In vitro interests exist. regeneration of garlic through callus culture. Journal of Biological Sciences. REFERENCES 2004;4(2):189–91. 13. Collins HA. Garlic and Cardiovascular Disease. In: Functional foods, diet, 1. Nair A, Khar A, Hora A, Malik CP. Garlic : cardiovascular disease and diabetes. Its importance and biotechnological Arnoldi A, Dodds F, (Eds.). Woodhead improvement. An International Journal of Publishings, U.K. 2004;240-295. Life Sciences. 2013;2(1):72–89. 14. Takasu J, Uykimpang R, Sunga MA, 2. Tapsell LC, Hemphill I, Cobiac L, Patch Amagase H, Niihara Y. Aged garlic extract CS, Sullivan DR, Fenech M. Health therapy for sickle cell anemia patients. benefits of herbs and spices: The past, the BMC Blood Disorders. 2002;2:3. present, the future. Medical Journal of 15. Haque MA, Nath UK, Ahmad QN, Alam Ausralia. 2006;185:1-24. MS. Effect of 2,4-D and BAP on in vitro 3. Moyers SB, Garlic in health, history, and regeneration of Garlic (Allium sativum). world cuisine. Suncoast Press; 1996. Online Journal of Biological Science. 4. Yeh YY, Liu L. Cholesterol-lowering effect 2003;2(12):771-774. of garlic extracts and organosulfur 16. Rahim MA, Amin MMU, Haider MA. Good compounds: Human and animal studies. quality seed cloves production technology The Journal of Nutrition. 2001;131(3): and its implications on bulb production of 989-993. garlic in Bangladesh. Allium Improvement 5. Sivam GP. Protection against Helicobacter Newsletter. 1993;3:42-46. pylori and other bacterial infections by 17. Lot H, Chovelon V, Souche S, Delecolle B. garlic. The Journal of nutrition. 2001; Effects of onion dwarf and leek yellow 131(3):1106-1108. stripe viruses on symptomatology and yield

9

Haider et al.; BBJ, 8(3): 1-12, 2015; Article no.BBJ.18619

loss of three French garlic cultivars. Biotechnology in Agricultural and Foresty Phytopathology. 1998;82:1381-1385. 39. Springer, Tokyo. 1997:3-19 18. Metwally EI, Denery ME, Dewir TH. 28. Ali AA, Metwally EI. Somaclonal variation Influences of explants type and enzyme as a source of variability in garlic breeding. incubation on isolated protoplast density In: Proceedings of the First Egyptian Italian and viability in two garlic cultivars. Pakistan Symposium on Biotechnology. Assiut. Journal of Botany. 2014;46:673-143. Egypt; 1992. 19. Robledo PA, Villalobos AVM, Jofre GAE. 29. Salam MA, Ali ME, Reza MSH, Islam S, Efficient plant regeneration of garlic (Allium Rahman SMM. Callus induction and sativum L.) by root tip culture. In vitro regeneration of indigenous garlic (Allium Cellular Developmental Biology. Plant. sativum L.). American Journal of Plant 2000;36:416-419. Physiology. 2008;3(1):33-39. 20. Kim EK, Hahn EJ, Murthy HN, Paek KY. 30. Murashige T, Skoog F. A revised medium High frequency of shoot multiplication and for rapid growth and bioassay with tobacco bulblet formation of garlic in liquid cultures. tissue cultures. Physiologia Plantarum. Plant Cell Tissue Organ Culture. 2003;73: 1962;15:473-497. 231-236. 31. Tiwari S, Tripathi MK, Khare UK, Pathak 21. Luciani GF, Mary AK, Pellegrini C, SK. Response of exogenous growth Curvetto NR. Effects of explants and regulators on callus proliferation and growth regulators in garlic callus formation morphogenesis on mature embryo culture and plant regeneration. Plant Cell Tissue of onion (Allium cepa L.). Journal of Organ Culture. 2006;87:139-143. Vegetation Science. 2004;31(2):107-111. 22. Xu Z, Um YC, Kim CH, Lu G, Guo DP, Liu 32. Nasim SA, Mujib A, Kapoor R, Fatima S, LH. Effect of plant growth regulators, Aslam J. Somatic Embryogenesis in Allium temperature and sucrose on shoot Sativum L. (Cv. Yamuna Safed 3): proliferation from the stem disc of Chinese Improving Embryo Maturation and jiaotou (Allium chinense) and in vitro Germination with PGRs and bulblet formation. Acta Physiologiae Carbohydrates. Anales de Biologia. 2010; Plantarum. 2008;30:521-528. 32:1–9. 23. Yanmaz R, Yazar E, Kantoglu KY, Alper A. 33. Xiao-Dong CAI, Gui-Yuan WANG, Wen- In vitro plant regeneration and bulblet Juan CAO. In vitro induction and formation of Tunceli garlic (Allium proliferation of callus from immature tuncelianum (Kollman) Ozhatay, Matthew, cotyledons and embryos of Juglans regia Siraneci) by shoot and root culture. Journal cv. ‘Xiangling’. Notulae Botanicae Horti of Food, Agriculture and Environment. Agrobotanici. 2013;41(2):378-384. 2010;8:572-576. 34. Zheng HR, Shen MJ, Zhong WJ, Znahg 24. Lee SY, Kim HH, Kim YK, Park NI, Park ZQ, Zhou Y, Zheng HR, et al. Induction SU. Plant regeneration of garlic (Allium and utilization of globular bodies on calli sativum L.) via somatic embryogenesis. from garlic (Allium sativum L.) leaf Scientific Research and Essay. 2009;4: explants. Study of cellular histology in 1569-1574. morphogenesis. Acta Agriculturae 25. Kellar ERJ, Senula A. Micropropagation Shanghai. 1998;14(1):33-38. and chryopreservation of garlic (Allium 35. Evenor D, Levi-Nissim A, Afgin L, Lilien- sativum L.). In: Lambardi M, et al., editors. Kipnis H, Watad AA. Regeneration of Protocols for micropropagation of selected plantlets and bulblets from explants and economically-important horticultural plants, callus of Allium aflatunense cultivars and methods in molecular biology, Springer, selection from indigenous Israeli Allium NY, USA. 2013;994:353-368. ampeloprasum. Acta Horticulture. 1997; 26. Metwally EI, Denery ME, Dewir TH, Naidoo 430:225-230. Y. In vitro propagation of garlic (Allium 36. Al-Zahim MA, Ford-Lloyd VB, Newsbury sativum L.) through adventitious shoot HJ. Detection of somaclonal variation in organogenesis. African Journal of garlic (Allium sativum L.) using RAPD and Biotechnology. 2014;13(38):3892-3900. cytological analysis. Plant Cell Report. 27. Nagakubo T, Takaichi, Oeda. 1999;18:473-477. Micropropagation of Allium sativum L. 37. Mohamed-Yasseen Y. In vitro shoot (garlic). In: Bajaj YPS, (Eds.). proliferation and plant regeneration from callus derived from inflorescence and

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Haider et al.; BBJ, 8(3): 1-12, 2015; Article no.BBJ.18619

flower of Allium ampeloprasum var. kurrat. porrum). Bulletin of the Faculty of Science Journal of Agricultural Science. 2001;9: of Cairo University. 2003;54:495-508. 387-396. 48. Barandiaran X, Martín N, Rodriguez CM, 38. Rębilas K, Rębilas A. Auxin concentrations Pietro DA, Martín J. An efficient method for control the average DNA content in cells of callus culture and shoot regeneration of in vitro cultures: A theoretical model and garlic (Allium sativum L.). HortScience. comparison to experimental data for Allium 1999;34:348-349. cepa and Allium sativum. Plant Cell Tissue 49. Mehta J, Sharma A, Sharma N, Megwal S, Organ Culture. 2008;95:89-99. Sharma G. International Journal of Pure & 39. Gantait S, Mandal N, Das PK. An overview Applied Bioscience An Improved Method on in vitro culture of genus Allium. for Callus Culture and In vitro Propagation American Journal of Plant Physiology. of Garlic (Allium Sativum L.). International 2010;5:325-337. Journal of Pure & Applied Bioscience. 40. Myers JM, Simon PW. Continuous callus 2013;1(1):1–6. production and regeneration of garlic 50. Scotton DC, Benedito VA, Molfetta JB, (Allium sativum L.) using root segments Ines B, Rodrigues FP, Tulmann-neto. from shoot tip-derived plantlets. Plant Cell Response of root explants to in vitro Report. 1998;17:726-730. cultivation of marketable garlic cultivars. 41. Toaima N, Novak E, Schumann G. Callus Horticultura Brasileira. 2013;31:80–85. induction from different explants of 51. Kizil S, Icgil DY, Khawar KM. Improved in commercial cultivars of leek, Allium vitro regeneration and propagation of ampeloprasum var. Porrum L. Acta tunceli garlic (Allium tuncelianum L.). Horticulture. 2003;597:303-309. Journal of Horticultural Science & 42. Zheng SJ, Henken B, Ahn Y, Krens F, Kik Biotechnology. 2014;89(4):408–11. C. The development of a reproducible 52. Haque MS, Wada T, Hattori K. High Agrobacterium tumefaciens transformation frequency shoot regeneration and plantlet system for garlic (Allium Sativum L.) and formation from root tip of garlic. Plant Cell the production of transgenic garlic resistant Tissue and Organ Culture. 1997;50:83-89. to beet armyworm (Spodoptera exigua 53. Haque MS, Wada T, Hattori K. Anatomical Hubner). Molecular Breeding. 2004;14: changes during in vitro direct formation of 293-307. shoot bud from root tips in garlic (Allium 43. Nagasawa A, Finer JJ. Induction of sativum L.). Plant Production Science. morphogenic callus cultures from leaf 1999;2:146-153. tissue of garlic. HortScience. 1998;23: 54. Haque MS, Wada T, Hattori K. Shoot 1068-1070. regeneration and bulb formation from 44. Schavemaker CM, Jacobsen E. shoot and root meristem of garlic cv. Development of a cyclic somatic Bangladesh local. Asian Journal of Plant embryogenesis regeneration system for Science. 2003;2(1):23-27. leek (Allium ampeloprasum L.) using 55. Seabrook JEA. In vitro propagation and zygotic embryos. Plant Cell Report. 1995; bulb formation in garlic. Canadian Journal 14:227-231. of Plant Science. 1994;74(1):155-158. 45. Hong W, Debergh P. Somatic 56. Suh SK, Park HG. Plant regeneration from embryogenesis and plant regeneration in the culture of garlic root explants. J. garden leek. Plant Cell Tissue Organ Korean Soc. HortScience. 1995;36(1): Culture. 1995;43:21-28. 31-37. 46. Silvertand B, Rooyen A, Lavrjsen PV, 57. Roksana R, Alam MF, Islam R, Hossain Harlem AM, Jacobsen EV. Plant MM. In vitro bulblet formation from shoot regeneration via organogenesis and apex in garlic (Allium sativum L.). Plant somatic embryogenesis in callus cultures Tissue Culture. 2002;12(1):11-17. derived from mature zygotic embryos of 58. Ramakrishnan M, Antony Ceasar S, leek (Allium ampeloprassum L.). Duraipandiyan V, Daniel MA, Ignacimuthu Euphytica. 1996;71:261-270. S. Efficacious somatic embryogenesis and 47. Mohammad-Yasseen Y, Nasr MI. Callus fertile plant recovery from shoot apex induction and plant regeneration from explants of onion (Allium cepa L.). In vitro kurrat (Allium ampeloprasum var. kurrat) Cellular & Development Biology; 2013. and leek (Allium ampeloprasum var. Plant. DOI:10.1007/s11627-013-9510-3.

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Haider et al.; BBJ, 8(3): 1-12, 2015; Article no.BBJ.18619

59. Haque MA, Sultana MS, Quddus T, Regeneration and Chromosomal Mousumi AM, Parvin MA, Mahmud MA. Manipulation. BAU Res. Prog. 1012;23:39. Improvement of Garlic through in vitro (Abstr.). ______© 2015 Haider et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Peer-review history: The peer review history for this paper can be accessed here: http://sciencedomain.org/review-history/10000

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