IJBPAS, December, 2018, 7(12): 1996-2004 ISSN: 2277–4998 ROSMARINIC ACID CONTENT OF GENETICALLY STABLE CLONES OF Ocimum americanum L. (LEMON BASIL) REGENERATED THROUGH IN VITRO SHOOT BUD MULTIPLICATION AND ROOT CULTURE BISWAS T*1 AND MUKHOPADHYAY S2 1*: Rishi Bankim Chandra College, P.O. Naihati, 24 Parganas (N), West Bengal, India, Pin 743165 2: Centre of Advanced Study, Department of Botany, University of Calcutta, Kolkata 700019, India *Corresponding author: Dr. Trayee Biswas: E Mail: [email protected]; Mob. No. 9433516357 th th th st Received 27 March 2018; Revised 15 April 2018; Accepted 19 Oct. 2018; Available online 1 Dec. 2018 https://doi.org/10.31032/IJBPAS/2018/7.12.4591 ABSTRACT Ocimum americanum L. is an annual herb with citrus scent therefore called lemon basil, reflects their high content of phenolic compounds including rosmarinic acid. In the present investigation an attempt has been made to establish in vitro shoot bud multiplication and root culture, for the maintenance of uniform progeny of the plant as a source of explant as well as rosmarinic acid production in a short period of time. Murashige and Skoog’s basal medium supplemented with 1.0 mg/l kinetin in combination with 0.2 mg/l α-napthaleneacetic acid (NAA) was found to be effective for inducing multiple shoots (27.4±0.534) from the apical bud explants. The shoots were transferred to ½ MS basal medium for rooting. Roots of in vitro regenerated plants were used to establish in vitro root cultures in liquid MS basal media of one fourth strength, supplemented with 1.0 mg/l NAA and 10.0 mg/l putrescine. Somatic chromosome analysis has indicated genomic stability of the regenerates. The increased level of rosmarinic acid content of the in vitro root cultures (2.45±0.06 % of dry weight) as compared to in vivo plants was recorded. Keywords: Genomic stability, Ocimum, Rosmarinic acid Abbreviations: MS - Murashige and Skoog’s basal medium, NAA - α -napthaleneacetic acid 1996 IJBPAS, December, 2018, 7(12) Biswas T*and Mukhopadhyay S Research Article INTRODUCTION Ocimum americanum L. commonly known establishment of root culture for production as lemon basil, is widely distributed in of rosmarinic acid since they show rapid tropical Africa and Asia [1]. O. growth and stable metabolic productivity americanum L. is morphologically without the influence of seasonal intermediate between O. canum and O. variations. basilicum. It is suggested that O. MATERIALS AND METHODS americanum arose as a natural hybrid Seeds of Ocimum americanum L. (lemon between the diploid O. canum (2n=24) and basil) were collected from Sutton and Sons the tetraploid O. basilicum (2n=48) (India) Pvt Ltd, Kolkata having accession followed by doubling of chromosomes number 16 35 58. The plants grown from (2n=72) [2]. It has been reported that these seeds were maintained in the rosmarinic acid is the predominant phenolic experimental garden, Department of compound found in basil accessions Botany, University of Calcutta, for further including lemon basil and is supposed to studies. Voucher specimens of the species act as a preformed constitutively are deposited in the herbaria of University accumulated defence compound [3]. The of Calcutta, (CUH, Kolkata) bearing biological activity of rosmarinic acid is accession number - West Bengal, Kolkata, described as antibacterial, antiviral, and Ballygunge Science College, Experimental antioxidative [4]. Rosmarinic acid content Garden, date 22.09.10, Biswas 005, Acc. no of basil varies greatly in wild population 200010 (CUH). Apical twigs (2.0 cm) from because of cross pollination, heterozygosity in vitro germinated seedlings (21 days after and seasonal variations. In addition, the germination) were used as explants for in pharmaceutical companies also make vitro shoot bud multiplication. Adventitious indiscriminate use of wild populations of roots (1.5-2.0 cm in length, 0.1 g fresh different medicinal plants for their own weight) excised from in vitro plants interests hence reducing the quality of regenerated through shoot bud active principle contents. The conventional multiplication, were used as explants for method for propagation of Ocimum root culture establishment. Seeds of O. americanum L. is seed germination and this americanum L. were first washed in plant cannot be vegetatively propagated. aqueous liquid detergent solution (Tween The present study describe establishment of 20, SRL; two drops in 100 ml) for 12 min uniform progeny of the plant through in at room temperature and rinsed thrice with vitro shoot bud multiplication and distilled water and were then treated with 1997 IJBPAS, December, 2018, 7(12) Biswas T*and Mukhopadhyay S Research Article an aqueous 0.1 % (w/v) HgCl2 solution in and each treatment was repeated for three order to establish maximum contaminant times in five replicates. Chromosome free cultures without affecting percentage analysis from root tip cells of in vitro of germination. Seeds were germinated in regenerates was carried out following vitro on Murashige and Skoog’s (MS) [5] propionic orcein staining method to modified basal medium supplemented with determine the somatic chromosome number 3% sucrose, 0.05 mg/l (w/v) ascorbic acid, of individual clones of each species [7,8]. and 0.1 mg/l (w/v) glutamine and 0.25% Dried plant tissues were used for extraction (w/v) Gelrite®. The shoot bud of phenolic compounds. 5.0 mg of dried multiplication was carried out using shoots and roots were pulverized in a modified MS basal medium supplemented mortar and suspended in 2.0 ml of aqueous with NAA (α-napthaleneacetate, C12H10O2) methanol (80:20 absolute methanol: water, and kinetin (6-furfurylaminopurine, w/v) and absolute methanol (100% C10H9N5O0). The research is conducted in methanol, HPLC grade) separately in factorial design based on Completely ambient conditions. Crude extract was Random Design (CRD) [6]. The factorial prepared for quantitative determination of levels of 5 treatments (0.0, 0.5, 1.0, 1.5, 2.0 total phenol and rosmarinic acid following and 2.5 mg/l) were repeated for three times the method of Ellis and Towers (1970) [9] in five replicates. The concentrations of and Didier and Pasquier (1994) [10]. The auxin (NAA) were 0.0 and 0.2 mg/l and crude extracts were weighed to calculate tested in case of all the 5 treatments. The the yield (g crude/100 g dried plant regenerated shoots were further cultured in material i.e. percentage) [11,12] and stored basal media for 4 weeks for further in a refrigerator (- 40C), until further use for elongation and after attaining a length of qualitative and quantitative estimation. more than 3.0 – 4.0 cm, were excised and Separation was achieved by using HPLC transferred individually to rooting medium. (Shimadzu, SPD-10A UV-Vis detector, LC- In this case, first factor was strength of MS, 10AD Liquid chromatograph) with a C18 full and half strength respectively and the column, (150 x 4.6 mm, Hypersil) of second factor was NAA (0.0 and 1.0 mg/l). particle size 5.0 µm. using water: Media used for in vitro root culture was acetonitrile (83:17 v/v) as mobile phase. same modified MS basal medium as in Ultraviolet detection was set at 330 nm. vitro regeneration. The treatments were Rosmarinic acid contents were calculated concentrations of NAA (1.0 and 2.0 mg/l) using a calibration curve (i.e., concentration and putrescine (0.0 and 10.0 mg/l) (2 x 2) 1998 IJBPAS, December, 2018, 7(12) Biswas T*and Mukhopadhyay S Research Article vs peak area) and expressed as percentage third subculture passage. In O. americanum of dry weight (% of dry wt) [13]. L. the half strength of MS basal medium Data were analyzed statistically following showed the highest numbers of shoots with one way ANOVA and two way ANOVA roots and the strength of medium have [14]. If the sample means differ more than significant effect on rooting as revealed the LSD (Least Significant Difference), it from the ANOVA test. The influence of was considered that the two were mineral concentration of culture medium significantly different (P≤0.05). Statistical on rooting can be attributed to the analyses were done using SPSS version participation of inorganic ions in processes 15.0 for Windows (SPSS Inc., USA). regulating hormonal balance [23]. The RESULTS & DISCUSSION cytological status of the regenerates of In the present investigation apical shoot these three species was verified using buds from germinated seedling have been somatic chromosome analysis and the found to be suitable explants for in vitro genomic stability was found to be shoot bud multiplication. There are various maintained. In vitro root culture was reports of utilizing different shoot bud successfully established in low nutrient morphogenesis in different species of concentration (1/4 MS medium) along with Ocimum [15, 16, 17, 18, 19, 20, 21, 22]. an optimum concentration of an auxin The earliest (4.0±0.01 days) shoot initiation (NAA 1.0 mg/l) which are critical as well as maximum multiplication fold determinants in controlling the growth of (27.4±0.534) obtained in the medium adventitious roots. In case of O. containing 0.2 mg/l NAA and 1.0 mg/l americanum L., maximum average kinetin (Fig 1). The number of shoot buds rosmarinic acid content 2.45±0.06 % of dry reduced beyond 1.0 mg/l level of kinetin. A wt obtained in liquid ¼ MS medium further increase in NAA concentration supplemented with 1.0 mg/l NAA and 10.0 caused callusing and hyper hydricity of mg/l putrescine. Two way analysis of regenerated shoots without increasing variance of observed data revealed multiplication fold. All the cultures were significant (P ≤0.05) influence of maintained up to 16 weeks until the concentration of NAA (1.0 and 2.0 mg/l) multiplication rate declined and despite the on average rosmarinic acid content (Table quantitative variation in number of shoot 2).