Impaginato 239 Adv. Hort. Sci., 2018 32(2): 239-248 DOI: 10.13128/ahs-22287 Somatic embryogenesis, biochemical alterations and synthetic seed development in two varieties of coriander (Coriandrum sativum L.) M. Ali, A. Mujib (*), N. Zafar, D. Tonk Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard University, New Delhi 110062, India. Key words: biochemical attributes, conversion frequency, Coriandrum sativum L., somatic embryogenesis, synthetic seeds. Abstract: Somatic embryogenesis (SE), biochemical alterations and synthetic seed formation were carried out in two Coriandrum sativum L. varieties (Rajendra Swathi ‘RS’ and Co-1). Callus was induced profusely in 1.0 mg/l 2,4- dichlorophenoxy acetic acid (2,4-D) added MS medium but Co-1 had more cal- lus induction frequency (96.0%) compared to RS (89.3%). The callus turned into embryogenic tissue and variable embryogenic frequency (77.6% in RS and 72.8% in Co-1) was noted. Somatic embryos started to differentiate on the same 2,4-D added medium but the numbers of somatic embryos were more in RS (63.0 embryos per culture) compared to Co-1 (51.0 embryos per culture). These somatic embryos progressed well and showed maximum maturation in RS (78.7%) in 0.25 mg/l 6-benzyladenine (BA) + 0.5 mg/l α-naphthalene acetic acid (NAA) added medium. The biochemical analyses of non-embryogenic-, embryogenic-callus and different stages of embryos were conducted in order to know the changes of physiology in different tissues. Sugar and proline content were noted to be high at embryo induction stage while protein level was higher at embryo maturation stage. Biochemical analysis also revealed that the cata- lase (CAT) and superoxide dismutase (SOD) activities were higher at maturation stage of embryos compared to other embryogenic stages. Matured somatic embryos were germinated in MS added with 1.0 mg/l BA + 0.5 mg/l gibberellic acid (GA3) in which 83.3% and 76.7% plantlet regeneration were noticed in RS and Co-1 respectively. Somatic embryos were encapsulated in various alginate and calcium chloride (CaCl2) solutions and were kept in different temperature regimes for varied periods. On regeneration medium, the encapsulated embryos germinated into plantlets; in 3% sodium alginate + 100 mM CaCl2, maximum plant regeneration (74.0% in RS and 70.6% in Co-1) was noted. The influence of low temperature on storage of synthetic seeds and their conver- sion into plantlets were also studied and we noted that the 4°C was the opti- mum temperature for synthetic seed conservation and plantlet regeneration compared to -20°C and 25oC temperature conditions. (*) Corresponding author: amujib3@yahoo.co.in Citation: ALI M., MUJIB A., ZAFAR N., TONK D., 2018 - Somatic embryogenesis, biochemical alterations and synthetic seed development in two varieties of coriander (Coriandrum sativum L.). - Adv. Hort. Sci., 32(2): 239-248 Copyright: © 2018 Ali M., Mujib A., Zafar N., Tonk D. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 10 October 2017 Accepted for publication 12 January 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(2): 239-248 240 1. Introduction Coriandrum sativum L. is an annual herb and belongs to the family Apiaceae. The plant is used as a spice and flavouring compound (Burdock and Carabin, 2009). The plant has potential in pharma- ceutical industry as it exhibits antimicrobial (Cao et al., 2012), antioxidant (Hashim et al., 2005), antidia- betic (Eidi et al., 2009), hepatoprotective (Samojlik et al., 2010) and antiarthritic properties (Rajeshwari et al., 2012). Plant tissue culture technology is widely used for large scale plant propagation, beside being used as an in vitro experimental model for studying cellular processes like cell division, differentiation and morphogenesis, all play important key role in somatic embryogenesis (SE) and plant development (Zimmerman, 1993). SE is a process in which somatic cells undergo morphological and metabolic changes under in vitro cultural conditions, acquire embryo- genic potency and later develop into somatic embryos (Feher et al., 2003). Somatic embryos mimic zygotic embryos in various ways and hence it has proved to be a good model for studying morpho- physiological, biochemical and molecular events dur- ing the course of embryogenesis (Dodeman et al., 1997). The fast identification of embryogenic tissue is very important as it plays a central role in marker- assisted selection (Dodeman et al., 1997). Beside morphology, the embryogenic tissues are often dif- ferentiated from non-embryogenic callus by various biochemical markers and these markers based selec- tion would be of immense value of SE based micro- propagation (Samar et al., 2011). Embryogenic tis- sues have the ability of producing embryos for an extended period of time without any genetic alter- ation, which requires efficient conservation protocol for long-term preservation of coriander (Murthy et al., 2008). Somatic embryos also have an important application in the formation of synthetic seeds and this artificial seed (an alternative to natural seed) technology may be exploited as a complementary method for in vitro production of plantlets (Reddy et al., 2012). It also promotes fast plantlet regeneration, facilitates germplasm exchange and conservation (Palanyandy et al., 2015). The synthetic seed technol- ogy with advantages like easy handling, conservation with germplasm exchange possibility is being widely used between national and international laboratories (Rai et al., 2009). Beside bipolar somatic embryos, other explants like unipolar micro-bulbs, rhizomes, protocorms, nodal cuttings and shoot buds are used for synthetic seed (Rihan et al., 2011; Sharma and Shahzad, 2012). Among the various natural and syn- thetic polymers available for encapsulation, sodium alginate is used more frequently because of its easy gelling properties, non-toxicity and low cost (Saiprasad, 2001). Different concentrations of sodi- um alginate ranging from 1.5 to 6.0% are used in encapsulation in different plant species Cacicus caro- ta (Latif et al., 2007), Spartina alterniflora (Utomo et al., 2008), Catharanthus roreus (Maqsood et al., 2012), Rinacanthus nasutus (Meena et al., 2013). In the present study, an efficient synthetic seed forma- tion protocol was optimized in C. sativum and their regeneration potential was described after in vitro storage at different storage conditions. We also dis- cussed SE and associated biochemical alterations in two varieties of C. sativum. 2. Materials and Methods Seed germination and cultural conditions Seeds of two varieties of C. sativum, Rajenda swathi (RS) and Co-1 were obtained from National Research Centre for Seed Spices (NRCSS) Ajmer, Rajasthan, India for this experimental study. The seeds were washed thoroughly under running tap water using cetrimide as detergent and surface disin- fection was made with 0.1% (w/v) HgCl2 for 2 min. The seeds were rinsed three times with sterilized double-distilled water before allowed to germinate on half strength MS medium (Murashige and Skoog, 1962) without plant growth regulators (PGRs). The basal medium was solidified by adding 8.0 g l-1 agar. The pH of the medium was adjusted to 5.7 using 1.0 M NaOH and 0.1 N HCl and was sterilised in an auto- clave for 15 min at 121°C. All the reagents were pre- pared using water supplied by a Milli-Q system (Billerica, Massachusetts, USA). The cultures were incubated at 25±2°C under 12-h photoperiod provid- ed by cool white fluorescent lamps (100 µmol m-2 s-1 PFD). Callus induction and somatic embryogenesis (SE) Callus was induced from hypocotyl explants of 10- day old germinated seedlings. Different concentra- tions (0.5-2.0 mg/l) of 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5-1.0 mg/l α- naphthalene acetic acid (NAA) were used for callus induction. After 4 weeks in induction medium, the callus was trans- ferred in fresh medium for induction of somatic Muzamil et al. - Somatic embryogenesis of two Coriander varieties 241 embryos. Somatic embryos were induced on same 2,4-D added callus-induction medium. For embryo maturation, MS was supplemented with NAA (0.5-1.5 mg/l) and 6-benzyladenine, BA (0.25-0.5 mg/l). The germination of somatic embryos was made by trans- ferring matured green embryos into MS, added with 0.5-2.0 mg/l BA and 0.2, 0.5 mg/l gibberellic acid (GA3). Biochemical analysis Proline, protein and sugar assay. Proline estima- tion was made according to Bates et al. (1973). About 0.05 g of callus was homogenized in 2.0 ml of 3.0% aqueous sulphosalicylic acid under cold condi- tions; the homogenate was filtered with Whatman filter paper (No. 1). The filtrate (1.0 ml) was added with 1.0 ml ninhydrin and 1.0 ml glacial acetic acid; the reaction mixture was incubated for 1 h at 100°C. The reaction was terminated in ice bath and 2.0 ml toluene was added to it. Proline content was mea- sured by spectrophotometric assay at 520 nm. The concentration of proline is expressed in mg per 1 g fresh weight. Protein estimation was made following Bradford et al. (1976). The homogenate of 0.25 g callus in 1.5 ml phosphate buffer (0.1 M, pH 7.0) under pre-cold condition was centrifuged at 104 rpm for 10 min and 1.0 ml supernatant was added with 0.5 ml trichloroacetic acid (10%). After centrifugation, the pellets were washed with acetone and dissolved in 1.0 ml of NaOH (0.1 N). To 1.0 ml of aliquot, 1.0 ml of Bradford reagent was added and optical density was measured at 595 nm The protein concentration was expressed in mg per 1 g fresh weight. Dey (1990) method was used for sugar estima- tion. Callus tissue 0.1 g was extracted twice with 90% alcohol at 60°C. Final volume of extract was made up to 10 ml by adding DDW. After mixing 0.5 ml of aliquot with 0.5 ml of 5% phenol, 1.0 ml of concen- trated sulphuric acid was added and cooled in air. The optical density was measured at 485 nm. The sugar concentration was expressed mg per 1 g fresh weight. Analysis of catalase (CAT), ascorbate peroxidise (APX) and superoxide dismutase (SOD) activity. Calli at different stages such as non-embryogenic, embryogenic tissue and different embryo stages (induction, proliferation and maturation) were homogenized in 2.0 ml of 0.1 M extraction buffer (0.1 M K-phosphate, 0.5 mM EDTA, 1.0 mM ascorbic acid, pH 7.5). After centrifugation at 104 rpm for 20 min, the supernatant was used for enzyme analysis. CAT activity was determined according to Aebi (1984) method by measuring a decrease in the absorbance at 240 nm of reaction mixture containing 1.0 ml of 0.5 M reaction phosphate buffer (Na-phos- phates, pH 7.5), 0.1 ml EDTA, 0.2 ml enzyme extract and 0.1 ml H2O2. The reaction was run for 3 min. One unit of enzyme determines the amount necessary to decompose 1.0 μM of H2O2 per min. CAT activity was calculated by using the co-efficient of absorbance at 0.036 mM-1 cm-1. The activity of CAT was expressed in EU mg-1 protein. The method developed by Nakano and Asada (1981) was used for APX activity. To the mixture of 1.0 ml sodium buffer (0.1 M, pH 7.2), 0.1 ml of EDTA and 0.1 ml of enzyme extract, 1.0 ml of 0.5 mM ascorbate were added and the reaction was run for 3 min at 25°C. The APX activity was estimated by moni- toring the decrease in absorbance due to the break- down of ascorbate by APX and was calculated by using the coefficient of absorbance 2.81 mM-1 cm-1. The activity of APX was expressed in EU mg-1 protein. The SOD activity was estimated following Dhindsa et al. (1981) method with slight modifications. Callus tissue (0.1 g) was homogenised in 2.0 ml of extrac- tion mixture (0.5 M phosphate buffer (pH 7.3), 3.0 mM EDTA, 1.0% (w/v) polyvinylpyrollidone (PVP), 1.0% (v/v) Triton X100) and centrifuged at 104 for 10 min. The SOD activity in the supernatant was assayed by adding 0.1 ml enzyme extract with 1.5 ml reaction buffer, 0.2 ml methionine, 0.1 ml each of 1.0 M NaCO3, 2.25 mM Nitro Blue Tetrazolium (NBT) solu- tion, 3.0 mM EDTA, riboflavin and 1.0 ml of Millipore H2O was taken in test tubes and was incubated under light for 10 min at 25°C. The absorbance at 560 nm, 50% reduction in colour is 1.0 unit and the enzyme activity was expressed in EU mg-1 protein. Synthetic seed preparation Mature green somatic embryos at the cotyle- donary stage were collected and suspended in a solu- tion of MS, added with different concentrations of sodium alginate (2%, 3% and 4%) for a few seconds and then dropped into the sterile aqueous solution of calcium chloride (CaCl2:75 mM, 10 mM and 125 mM) in order to encapsulate the embryo. Encapsulated embryos in CaCl2 solution were shaken in an orbital shaker at 60 rpm. CaCl2 solution was poured off and the beads were washed twice with sterilized water and placed on sterilized filter paper to remove excess water. Adv. Hort. Sci., 2018 32(2): 239-248 242 Storage and conversion of synthetic seeds Encapsulated somatic embryos were stored at 4°C (in refrigerator), 25°C (incubation room temperature) and at -20°C for varied periods (weeks) in order to evaluate the viability and regeneration/conversion potential of stored synthetic seeds. The synthetic seeds were stored using airtight dark 100 ml conical flasks. The synthetic seeds were periodically removed from the respective storage temperatures and cul- tured in MS added with various plant growth regula- tors (PGRs). The conversion rate was recorded by observing the development of shoots in 1.0 mg/l BA and 0.5 mg/l GA 3 added MS after 2 and 4 weeks of culture. Statistical analysis For callus induction, hypocotyls explants were cul- tured in test tubes with a minimum of 30 explants per experiment. In case of somatic embryo, induction each experiment was replicated three times. Data on frequency of response and numbers of somatic embryos induced per 500 mg of callus were recorded after 4 weeks of culture. The data presented as mean and its standard deviation (mean ± SD). The signifi- cance of differences among means was carried out using Duncan’s Multiple Range Test, DMRT (Duncan, 1995) at P= 0.05. 3. Results Callus induction and somatic embryogenesis On 2,4-D (0.5-2.0 mg/l) supplemented MS medi- um, prolific callus was induced from hypocotyl explants. The optimum callus induction frequency of 96.0% and 89.3% was noticed in Co-1 and RS respec- tively with 1.0 mg/l 2,4-D added medium. The callus induction frequency decreased as the concentration of 2,4-D above 1.0 mg/l was used (Table 1). Callus induction was also observed on NAA at 0.5 mg/l added MS but the induced callus was non-embryo- genic in nature. The 2,4-D induced embryogenic cal- lus was white and friable, which produced globular embryos on the same induction medium (Fig. 1 a). Of the different 2,4-D concentrations used, 1.0 mg/l exhibited higher embryogenic ability (77.6%) in RS with 63.3 somatic embryos; in Co-1, the embryogenic ability was also equally high (72.8%) with relatively lower numbers of embryos (51.0) (Fig. 1 b). Beside 2,4-D, other PGR combinations were also tested for embryo differentiation. In 0.5 mg/l NAA + 0.25 mg/l BA amended medium, maximum differentiation of embryos (78.7% in RS and 74.0% in Co-1) was noticed (Fig. 1 c-d) and in increasing NAA concentrations the embryo differentiation decreased. The somatic Table 1 - Effect of different concentrations of 2,4-D on callus induction and somatic embryogenesis from hypocotyl explants of ‘Rajendra Swathi’ and ‘Co-1’ varieties of Coriandrum sativum Values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p= 0.05) according Duncan’s multiple range test. 2,4-D (mg/l) Rajendra Swathi Co-1 Callus induction (%) Embryogenic callus induction frequency No. of embryos formed/culture (0.5 g) Callus induction (%) Embryogenic callus induction frequency No. of embryos formed/culture (0.5 g) 0.5 86.0±4.0 a 55.3±2.5 b 39.3±2.1 b 89.3±3.0 b 52.0±2.64 b 36.3±2.08 c 1 89.3±4.2 a 77.6±3.2 a 63.0±4.5 a 96.0±2.3 a 72.8±3.0 a 51.0±2.64 d 1.5 74.0±4.0 b 58.0±2.6 b 44.5±2.5 b 80.7±4.1 c 54.2±2.51 b 41.5±2.0 b 2 47.3±1.1 c 45.2±2.3 c 31.0±3.0 c 64.0±2.0 d 42.0±3.0 c 27.0±3.0 d Fig. 1 - Somatic embryo formation in Coriandrum sativum (RS). a) Globular embryos at induction stage; b) Somatic embryos at proliferation stage; c and d) Somatic embryos at maturation stage (scale bar, a-2 mm; b-c= 3 mm; d=1 cm). Muzamil et al. - Somatic embryogenesis of two Coriander varieties 243 embryo germination was also influenced by PGRs combination; 1.0 mg/l BA + 0.5 mg/l GA3 showed maximum germination of embryos in both varieties (83.3% in RS and 76.7% in Co-1). The increased levels of BA reduced plantlet conversion rate (Table 2). Biochemical analysis The embryogenic callus is often biochemically dif- ferent from non-embryogenic tissues which could be used in marker assisted early selection. Protein, pro- line and sugar level were noted to be high in early embryogenic tissue compared to non-embryogenic callus (Fig. 2 a-c). The sugar content was also noted high at induction stage of embryogenesis (37.2 mg g m-1 fresh weight in ‘RS’ and 33.4 mg g m-1 fw in ‘Co- 1’) as compared to other stages of embryo (matured) and non-embryogenic callus. The same was the case with proline where induction stage had more levels of proline in both the varieties (2.78 mg gm-1 fw in RS and 2.54 mg g m-1 fresh weight in Co-1). Protein con- tent was higher at maturation stage of embryos showing 7.3 mg gm-1 fw in ‘RS’ and 7.0 mg gm-1fw in Co-1compared to other stages of tissues. Antioxidant enzymes activity behaved differently in different cultivating tissues (Fig. 2 d-f). The highest CAT activity was observed in RS with 5.4 EU mg-1 pro- tein min-1 compared to Co-1 with 4.7 EU mg-1 protein min-1 at matured stage of somatic embryos; so was SOD activity (4.8 EUmg-1protein min-1 and 4.3 EU mg-1 protein min-1 in RS and Co-1 respectively). The APX activity was highest at induction stage of embryos with 2.1 EU mg-1 protein min-1 in RS and 1.8 EU mg-1 protein min-1 in Co-1 compared to other tissues. Synthetic seed preparation and plantlet conversion Green and matured somatic embryos were encap- sulated in different gelling conditions. Good synthetic seeds were produced in combination of sodium algi- nate and calcium chloride mix, which later responded differently in germination medium (Fig. 3 a-c). The study revealed that 3% sodium alginate + 100 mM CaCl2 was the ideal mixture condition that had fairly good conversion rate (74.0% in RS and 70.6% in Co-1) compared to 2% (48.6% in RS and 43.3% in Co-1) and 4% (30.0% in RS and 26.6% in Co-1) sodium alginate solution (Table 3). The higher sodium alginate level (4%) reduced synthetic seed conversion ability. Similarly, the level of CaCl2 was also equally impor- tant in making synthetic seeds and later in obtaining Table 2 - Somatic embryo differentiation and germination frequency in ‘RS’ and ‘Co-1’ on different concentrations of NAA, BA and GA3 supplemented MS medium Values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p = 0.05) according Duncan’s multiple range test. NAA (mg/l) BA (mg/l) GA 3 (mg/l) Rajendra Swathi Co-1 Embryo differentiation Conversion rate Embryo differentiation Conversion rate 0 0.5 0.25 0 54.6±3.0 c 0 51.3±3.0 c 0 1 0.25 0 68.0±3.4 b 0 64.0±3.4 b 0 1 0.5 0 83.3±4.6 a 0 76.7±4.1 a 0 1.5 0.5 0 63.6±3.0 b 0 54.0±2.0 c 0.5 0.25 0 78.7±4.1 a 0 74.0±4.0 a 0 1 0.25 0 61.3±3.0 b 0 55.3±3.0 b 0 1 0.5 0 56.0±3.4 b 0 47.2±2.3 c 0 1.5 0.5 0 39.3±3.0 c 0 34.0±2.0 d 0 Fig. 2 - Biochemical and enzyme activities in two varieties of Coriandrum sativum (RS and CO-1). a) Proline content; b) Protein content; c) Sugar content; d) Catalase activity; e) Ascorbate peroxidase activity and f) Superoxide dismuta- se activity. Adv. Hort. Sci., 2018 32(2): 239-248 244 plantlets; 100 mM CaCl2 had a high plantlet conver- sion, followed by 75 mM and 125 mM. The synthetic seeds prepared at low (2%) sodium alginate +75 mM CaCl2 were soft, fragile and showed poor conversion; while high (4%) level of sodium alginate and CaCl2 (125 mM) produced hard beads, reducing conversion ability. The duration of CaCl2 exposure also had an influence on conversion; 20 min exposure resulted in higher rate of germination compared to 10 and 30 min exposure (data not shown). The synthetic seeds containing somatic embryos, were kept in three different storage temperatures for varying periods in order to find the right temperature for preservation. It was observed that the storage temperature and duration had a direct influence on synthetic seed viability and final conversion rate (Table 4). The encapsulated somatic embryos stored at 4°C for a week, showed higher regeneration ability (62.0% in RS and 58.6% in Co-1) compared to incuba- tion at room temperature (25oC) and -20oC where the conversion rate was 42.6% and 13.3% in RS and 34.6% and 9.3% in Co-1 respectively. The conversion rate decreased as the storage duration increased as was observed in RS (44.0%) and Co-1 (37.3%) after 5 weeks of 4oC storage (Table 4). The synthetic seed’s viability was lost in both the varieties when stored at ultralow temperature (-20oC) for extended period (3 weeks). The regeneration ability gradually declined with increasing storage time in all tested conditions. The plantlets derived from synthetic seeds had a prominent shoot primordia without much root growth and these shoots were transferred to root induction medium, added with 0.5 mg/l IBA. Well rooted plants (Fig. 3 d) were taken out from the test tube, washed with water and transferred in paper cup (Fig. 3 e). After 2 weeks of hardening, the cups were transferred to the field. Out of 80 plants trans- ferred to soil, 43 (55%) survived in the field. The sur- vived plants in outdoor looked healthy and morpho- logically similar to mother plants. 4. Discussion and Conclusions In these two varieties of coriander, callus was vig- orously induced from hypocotyl in 1.0 mg/l 2,4-D added MS medium, consistent with previous studies in which 2,4-D played a crucial role in callusing in var- Fig. 3 - Synthetic seed development and plantlet formation in Coriandrum sativum (RS). a) Encapsulated somatic embryos; b) Synthetic seeds on the germination medium; c) Germinating synthetic seeds; d) Rooted plantlet and e) Synthetic seed derived plant, grown in outdoor condition (scale bar, a= 1.5 cm, b-d= 1 cm, e=2 cm). Table 3 - Effect of different concentrations of sodium alginate and calcium chloride on the conversion rate of encapsulated somatic embryos on 1.0 mg/l BA and 0.5 mg/l GA3 MS medium Values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p= 0.05) according Duncan’s multiple range test. Alginate (%) Calcium chloride (mM) Conversion rate (%) Rajendra Swathi Co-1 2 weeks 4 weeks 2 weeks 4 weeks 2 75 36.7±2.3 d 45.3±3.0 d 29.3±2.3 d 38.0±3.5 d 100 42.6±3.0 c 48.6±2.3 cd 36.6±3.0 c 43.3±3.0 c 125 30.0±2.0 e 37.3±3.0 e 24.0±2.0 e 32.6±2.3 e 3 75 46.0±3.4 c 52.0±3.4 c 41.3±3.0 e 51.3±3.0 b 100 63.3±4.2 a 74.0±4.0 a 64.0±3.4 a 70.6±4.1 a 125 52.0±3.4 b 57.3±2.3 b 45.3±2.3 b 54.7±3.0 b 4 75 21.3±2.3 f 24.6±3.0 g 18.0±2.0 f 24.0±2.0 f 100 25.3±3.0 ef 30.0±2.0 f 21.3±2.3 e 26.6±2.5 f 125 12.0±2.0 g 20.6±2.3 g 10.6±1.15 f 19.3±1.1 f Muzamil et al. - Somatic embryogenesis of two Coriander varieties 245 ious investigated plants (Kim et al., 2011). In the pre- sent study, embryogenic callus started to differenti- ate somatic embryos in 2,4-D added medium but the continuous presence of 2,4-D inhibited somatic embryo development beyond globular/heart shaped stage. The formation of somatic embryos on callus induction medium was earlier reported in several other species (Kim et al., 2011). The hindrance of embryo differentiation and progression in continuous presence of 2,4-D has earlier been reported in a number studied cases and the transfer of embryo- genic tissues or embryos into ‘2,4-D-free’ or less PGR concentrated medium has been suggested for embryo development (Junaid et al., 2006). The embryogenic tissues with developing somatic embryos were later cultured on NAA and BA added medium in which fast embryo development and mat- uration were observed. Somatic embryo differentia- tion and maturation in NAA and BA added medium was earlier reported in several investigated plants (Junaid et al., 2006; Puhan and Rath, 2012). The use of sucrose, ABA and GA3 has also been noted to be very efficient in promoting somatic embryo develop- ment and maturation (Yang et al., 2013; Mujib et al., 2014). As the non-embryogenic callus transforms into embryogenic tissues, biochemical profiles also alter simultaneously. In this study, we noted differences in biochemical and enzyme activities in embryogenic and non-embryogenic tissues. Here, we noted increased protein and decreased soluble sugar levels in maturation stage of somatic embryo, which is in confirmatory with Kumar and Kumari (2010). Enhanced protein content was also observed here during maturation stage of embryo and it could be used as a good tissue- specific indicator. The increase in biochemical attributes in embryogenic callus over non-embryogenic tissue was previously reported in several plants (Nieves et al., 2003). The extra proline accumulation has been noted in response to stress, which is believed to act as osmo-regulator in protect- ing cells against osmotic perturbation (Elmaghrabi et al., 2013). Similar changes in biochemical attributes were earlier observed during transition to differenti- ated embryogenic state from meristematic tissue under in vitro cultural conditions (Samar et al., 2011). Kumar et al. (2010) observed alteration of enzyme activities during cellular differentiation, this and simi- lar other findings are important in understanding the metabolic changes in developmental events (Santos et al., 2011). Here, the enzyme activities were lower in non-embryogenic callus compared to embryogenic one, but it was higher at specific embryo stages. In Hevea brasiliensis similar increased peroxidase activi- ty was noted during embryo organization (Blanc et al., 2002). Increased APX and CAT activity in embryo- genic tissue, designates its higher efficiency in scav- enging H2O2 during SOD metabolism, prevents mem- brane lipids peroxidation process (Niknam et al., 2006). Beside distinguishing morphogenic events, CAT facilitates in forming resistant cell walls and helps in defense mechanism against reactive oxygen species (Gaspar et al., 2002). The induced mature somatic embryos were encapsulated in gelling mix and the conversion of synthetic seeds into plantlets was monitored. Singh et al. (2010) observed that the quality bead forma- tion and germination ability of synthetic seeds are partly dependent on concentrations of sodium algi- nate and calcium chloride solution and on duration of exposure. Our observation indicated that the somatic embryos, encapsulated in 3% sodium alginate + 100 Storage temperature Storage duration (weeks) Regeneration (%) Rajendra Swathi Co-1 2 weeks 4 weeks 2 weeks 4 weeks -20°C 1 12.6±2.3 f 13.3±1.1 f 6.6±1.1 f 9.3±1.15 e 3 0 0 0 0 1 57.3±4.1 a 62.0±4.0 a 54.0±3.5 a 58.6±3.0 a 4oC 3 38.0±2.0 b 44.0±3.4 b 31.3±3.0 b 37.3±3.0 b 5 24.0±2.0 d 32.0±2.0 c 19.3±2.3 d 27.3±3.0 c 7 10.6±1.1 f 18.6±1.1 e 09.3±1.1 f 16.0±2.0 d 25°C 1 33.3±3.0 c 42.6±3.0 b 27.3±2.3 c 34.6±2.3 b 3 18.0±2.0 e 27.3±2.3 d 13.3±1.15 e 19.3±1.15 d Table 4 - Conversion rate at temperature conditions of somatic embryos encapsulated in 3% sodium alginate and 100 mm CaCl2, after storage. MS was added with 1.0 mg/l BA and 0.5 mg/l GA3 Values are expressed as mean standard deviation, mean values within a column followed by different letters are significantly different (at p= 0.05) according Duncan’s multiple range test. Adv. Hort. Sci., 2018 32(2): 239-248 246 mM CaCl2 produced quality bead with more conver- sion as compared to the use of lower (2%) and higher level (4%) of sodium alginate. The earlier reports sug- gested that somatic embryos encapsulated with 3% sodium alginate and 100 mM CaCl2 produced uniform and moderately hard beads with optimum regenera- tion potential (Sarmah et al., 2010). The maximum plantlet generation frequency with 3% sodium algi- nate was found in Paulownia elongata (Gozukirmizi, 2003), Coelogyne breviscapa (Mohanraj et al., 2009), Stevia rebaudiana (Ali et al., 2012). In Stevia rebaudi- ana, hard beads were formed at 4% sodium alginate, which adversely affected germination rate (Andlib et al., 2011). The 20 min exposure in CaCl2 solution improved conversion of plantlets, the conversion rate however, declined with increasing exposure time and this observation corroborates with similar other pre- vious studies (Malabadi and Van Staden, 2005). Over duration of exposure affects germination ability of the synthetic seeds and this decline of conversion may be due to growth inhibition caused by over absorption and penetration of calcium chloride (Malabadi and Van Staden, 2005). The use of liquid MS for preparing gel matrix was found to be benefi- cial in emerging shoot and this may be attributed to the presence of nutrients in gel matrix, which appar- ently serves as a nutrient bed around the propagules and facilitates synthetic seed survival and germina- tion (Ara et al., 1999). Matrix of synthetic seed mim- ics endosperm of natural seed and this nutrient rich artificial endosperm is essential for maintaining sur- vival of germplasm (Antonietta et al., 1999). The stor- age conditions also affect the germination potential of synthetic seeds. Our observation indicated that the viability and regeneration ability of synthetic seeds decreased with increasing storage duration. The same view i.e. the longer storage of synthetic seeds reduced conversion ability was reported in ear- lier few cases (Parveen and Shahzad, 2014). During long storage, carbohydrate reserves decrease gradu- ally, which may be responsible for the reduced con- version ability of synthetic seeds (Ding et al., 1998). The storage of in vitro grown tissues at -20°C showed a very poor regenerative ability and loss of tissue via- bility. It is believed that the short spell of low tem- perature exposure helps forming ice crystals in tis- sues, prevents food exchange, causes frost injury/stress and reduces tissue viability (Mittler, 2006). The successful regeneration system requires ger- mination of synthetic seeds/mature somatic embryos into plantlets at fast pace. The encapsulated somatic embryos were germinated into plantlets on BA and GA3 added MS. The combination of BA and GA3 was found to be very responsive for somatic embryo ger- mination in Solanum lycopersicum (Godishala et al., 2011). The well-developed plantlets were transferred to IBA added half MS for promoting root growth. The positive effect of IBA on root induction was reported in several investigated plants like Camellia nitidissima (Lu et al., 2013), Talinum triangulare (Swarna and Ravindhran, 2013). The synthetic seed derived plants were healthy and morphologically very similar to par- ent plants. The developed plant regeneration proto- col from synthetic seeds will be very important in preserving coriander germplasm for short and medi- um term basis. In summary, synthetic seed development and plant regeneration after storage may provide a good alternative strategy for coriander germplasm. The ‘short and medium term’ storage can open up and enable conservation possibility of elite important coriander. 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