Appl Biochem Biotechnol (2014) 173:2267–2278 DOI 10.1007/s12010-014-1032-x

Improved Protocol for Somatic Embryogenesis and Calcium Alginate Encapsulation in Anethum graveolens L.: A Medicinal Herb Richa Dhir & G. S. Shekhawat & Afroz Alam

Received: 4 March 2014 / Accepted: 19 June 2014 / Published online: 29 June 2014 # Springer Science+Business Media New York 2014

Abstract An improved procedure has been developed for efficient somatic embryogenesis in Anethum graveolens. Green friable embryogenic callus was obtained from hypocotyl segments on medium augmented with 2,4-dichlorophenoxyacetic acid (2,4-D). The highest embryogenic callus induction frequency of 87 % was obtained on Murashige and Skoog (MS) medium containing 1.13 μM 2,4-D. At lower concentration of 2,4-D (0.34 μM) callus turned dark in color and slow growing. Embryogenic cultures (76 %) responded with a mean number of 43 globular and 18 heart stage embryos. Somatic embryo maturation and subsequent conversion into plantlets took place on MS lacking growth regulators. Maximum number of somatic embryos developed on MS medium was 128.3 (per flask) and a plantlet conversion of 82 % was observed. Calcium alginate beads were produced by encapsulating somatic embryos. Highest percent germination (83 %) was observed on 0.8 % agar solidified MS medium with the plantlets acquiring an average length of 2.1 cm. Encapsulated somatic embryos could be stored at 4 °C up to 60 days with a conversion frequency of 49.3 %. Highest protein and proline content has been observed in embryogenic callus with small globular embryos. During morphological differentiation of the somatic embryos, changes in the antioxidant enzymatic system were observed. Superoxide dismutase (SOD) activity increased during initial stages and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) activities were detected. Keywords Anethum graveolens L . Antioxidative enzymesm . Encapsulation . Somatic embryogenesis Abbreviations

2,4-D APX

2,4-Dichlorophenoxyacetic acid Ascorbate peroxidase

R. Dhir : A. Alam Department of Bioscience and Biotechnology, Banasthali University, Rajasthan 304022, India G. S. Shekhawat (*) Department of Botany, Jai Narain Vyas University, Rajasthan 342001, India e-mail: [email protected]

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CAT MS NAA POD SE SOD

Appl Biochem Biotechnol (2014) 173:2267–2278

Catalase Murashige and Skoog α-Naphthalene-acetic acid Peroxidase Somatic embryogenesis Superoxide dismutase

Introduction Anethum graveolens L. commonly known as Dill is an annual aromatic herb belonging to the genus Anethum and the family Apiaceae. The plant has feathery leaves and bares tiny yellow flowers. The foliage and fruits of this plant are extensively used as culinary herb and spice in foods for their flavor, aroma, and preservative properties. Members belonging to Apiaceae are important ingredients of many herbal products [1]. The plant has various pharmacological effects such as antimicrobial [2], antihyperlipidaemic, and antihypercholesterolaemic [3] and is an important ingredient in various ayurvedic preparations [4]. Additionally, in folklore, Dill is used as a cure against a range of gastrointestinal disorders, e.g., flatulence, indigestion, colic pain, etc. The plant is cultivated particularly for its characteristic volatile compounds. Caravone and limonene are main constituents of dill seed oil whereas α-phellandrene, dill ether, and myristisin are important for odor of dill herb [5]. Eighty tons of weed oil and 70 tons of seed oil is produced worldwide from Anethum [6]. Low seed set is a serious problem which has been witnessed in members of Apiaceae. Lack of proper pollination and fertilization, occurrence of male flowers, and underdeveloped flowers are some reasons for low seed set in Apiaceae [7]. Innovations in biotechnological and tissue culture methods have paved new avenues for large-scale propagation of such plants to meet their industrial demands [8]. Development of embryos from somatic cells is referred to as somatic embryogenesis [9]. Somatic embryogenesis leads to the development of a functional meristem and a vascular system where lies the significance of somatic embryogenesis (SE) when compared to the other modes of regeneration. Another significance of SE is that a single step is required for the development of the root/shoot axis [10]. Improved protocols to achieve maximum production of useful plants with concomitant reduction in cost are being designed constantly. A few attempts have been reported on somatic embryogenesis in A. graveolens [11, 12]. However, further research in this area is prerequisite to develop this technique in direction of higher SE efficiency and easier tissue culture procedures to make the latter practical and convenient. The rationale behind this study was to establish a procedure for high-frequency somatic embryogenesis in Dill, encapsulation of SEs for short-term storage, and evaluation of the role of antioxidant enzymes in the course of SE development. To the best of our acquaintance, development of alginate encapsulated beads has been reported for the first time in A. graveolens.

Material and Methods Plant Material and Disinfection A. graveolens seeds were obtained from Jawaharlal Nehru Krishi Vishwa Vidyalaya, Madhya Pradesh, India, and were cultivated in the botanical garden of the Faculty of Bioscience & Biotechnology, Banasthali University. The seeds were kept under running water for 20 min.

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Then immersed in a few drops of detergent (Tween-20) and 20 % commercial bleach (4 % sodium hypochlorite) for 15 min, rinsed with sterile distilled water, and planted in conical flasks (100 ml; Borosil, India) containing 50 ml ¼ strength Murashige and Skoog (MS) medium. Basal Medium and Culture Conditions MS basal medium [13] supplemented with sucrose (3 %w/v) and agar (0.8 %w/v) was prepared. Prior to autoclaving the medium, pH was adjusted to 5.8. Conical flasks with 50 ml of medium were capped using plugs made of non-absorbent cotton. The medium was autoclaved at 121 °C and 1.06 kg cm−2 for 15 min. All cultures were incubated at 25±2 °C temperature and 50±5 % humidity in a culture room with 16-h photoperiod (irradiance 40 μmol m−2 s−1). Callus Induction and Somatic Embryogenesis Hypocotyl explants obtained from in vitro-germinated seedlings were used to establish callus cultures. Explants were placed horizontally on MS medium supplemented with 2,4-D (0.23– 2.26 μM) or NAA (0.26–2.68 μM) for callus induction. Percentage response (%), growth of callus, fresh weight, and morphology of callus were recorded after 4 weeks of culture. For maintenance and multiplication, the calli were subcultured on MS medium augmented with 1.13 μM 2,4-D. Thereafter, the obtained calli were transferred to lower concentrations (0.23 and 0.34 μM) of 2,4-D to bring about SE. Clumps of embryogenic calli on which globular embryos were observed were divided into parts (approximately 10 mg) and transferred to MS medium lacking growth regulators for SE development and maturation. Calcium Alginate Encapsulation Torpedo stage embryos measuring approximately 3–4 mm in size were used for calcium alginate encapsulation. For encapsulating embryos, sodium alginate (Hi-Media, Mumbai, India) was prepared in liquid MS medium, concentration ranging from 1 to 5 % (w/v) and calcium chloride (75 and 100 mM) was made in double-distilled water. Sodium alginate was sterilized and SEs were suspended in this gelling mixture, thereafter dispensed drop wise into the CaCl2 solution. Further, to accomplish the ion-exchange required for the formation of beads, they were kept in CaCl2 solution for 20–30 min. On hardening, resultant beads were washed in sterilized distilled water. To get rid of excess water, encapsulated embryos were placed on filter paper. Encapsulated SEs were stored at 4 °C in dark up to 60 days for further experiments. Encapsulated SEs were placed on different planting medium (¼,-, ½-, and fullstrength liquid and 0.8 % agar-solidified MS medium) for germination. Storability of both encapsulate and non-encapsulated SE was checked periodically (0, 15, 30, 45, and 60 days) by placing them on full-strength MS medium. Acclimatization and Field Transfer Plantlets obtained from somatic embryos and synthetic seeds were planted in small plastic cups after removing agar from the roots by washing under running tap water. The potting mixture consisted of a 1:1:1 ratio of sterile soil, sand, and vermiculite. Plantlets were enclosed with perforated polythene bags to maintain high humidity. After 2 weeks, polythene membranes were opened and plants were placed in field under a normal day-length regime.

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Histological Examination SEs were fixed in ethyl alcohol, formalin, and glacial acetic acid (90:5:5v/v). After fixation, tissue was dehydrated in graded ethanol series. The dehydrated tissue was embedded in paraffin wax. Sections (10 μm thick) were cut using a hand microtome (Coslab, India). The cut sections were mounted on glass slides and stained with 0.05 % toluidine blue prepared in phosphate buffer solution containing citric acid (pH 6.8) [14]. Biochemical Estimation Callus samples were acquired from 4-week-old cultures (E1), embryogenic callus on induction medium with small globular embryos were obtained from 7-week-old cultures (E2) and cultures with mature embryos were obtained from 9-week-old cultures (E3). Total Protein, Proline, and H2O2 Content Total protein content was determined following the method described by Lowry et al. [15]. Of the samples, 0.1 g were extracted in 0.01 M phosphate buffer pH 7.4. Absorbance was taken at 700 nm. The amount of proline was estimated by the method of Bates et al. [16]. Of the tissue, 0.3 g was used for the purpose and absorbance was recorded at 520 nm. H2O2 content was estimated using the method of Alexieva et al. [17]. After developing the reaction mixture for 60 min in the dark, absorbance was read at 390 nm. Antioxidant Enzyme Activity For enzyme extraction, 0.2 g of sample was homogenized in 3 ml of extraction buffer (50 mM phosphate buffer pH 7.8; 0.2 mM EDTA; 1 % PVP) in a prechilled mortar and pestle. However, for estimation of ascorbate peroxidase activity, 1 mM ascorbate was incorporated in the extraction buffer. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was used for estimation of antioxidant enzyme activities. Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by the method of Beauchamp and Fridovich [18]. Ascorbate peroxidase activity (APX; EC 1.11.1.7) was estimated according to Chen and Asada [19]. Catalase (CAT; EC 1.11.1.6) activity was estimated by the method of Aebi [20]. Analysis of peroxidase (POD; EC 1.11.1.7) capacity was based on the method of Chance and Maehly [21]. Statistical Analyses For all the concentrations used in the study, 15 replicates were examined. Data were recorded for three independent experiments and represented as mean ± SE. For all the biochemical parameters, data were collected from three replicates and expressed as the mean ± SE. Data were analyzed for one-way analysis of variance (SPSS version 16). Significance of difference (P

Improved protocol for somatic embryogenesis and calcium alginate encapsulation in Anethum graveolens L.: a medicinal herb.

An improved procedure has been developed for efficient somatic embryogenesis in Anethum graveolens. Green friable embryogenic callus was obtained from...
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