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Allium Sativum) Accessions of Bangladesh

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Allium Sativum) Accessions of Bangladesh British Biotechnology Journal 8(3): 1-12, 2015, Article no.BBJ.18619 ISSN: 2231–2927 SCIENCEDOMAIN international www.sciencedomain.org In vitro Plantlet Regeneration of Four Local Garlic (Allium 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 Plant 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 _____________________________________________________________________________________________________ *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 Allioideae under family Amaryllidaceae. 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 plants 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 2 Haider et al.; BBJ, 8(3): 1-12, 2015; Article no.BBJ.18619 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].
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