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Industrial Crops and Products 50 (2013) 468– 477

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l h om epa ge: www.elsev ier .com/ locate / indcrop

n improved in vitro encapsulation protocol, biochemical analysis andenetic integrity using DNA based molecular markers in regeneratedlants of Withania somnifera L

igar Fatimaa,c, Naseem Ahmada, Mohammad Anisa,b,∗, Iqbal Ahmadc

Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202 002, IndiaDepartment of Plant Production, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi ArabiaDepartment of Agricultural Microbiology, Aligarh Muslim University, Aligarh 202 002, India

r t i c l e i n f o

rticle history:eceived 8 April 2013eceived in revised form 20 July 2013ccepted 2 August 2013

eywords:ynthetic seedsodal segmentscclimatizationntioxidant enzymesAPD

SSR

a b s t r a c t

Non-embrogenic, synthetic seeds were produced by encapsulating nodal segments (containing axil-lary buds) of Withania somnifera L. in calcium alginate hydrogel containing Murashige and Skoog (MS)medium. A 3% sodium-alginate with 100 mM calcium cloride found to be the optimum concentrationfor the production of uniform synthetic seeds. Effect of different treatments (M1–M5), i.e. MS mediumcontaining different concentrations of cytokinins (0.5, 1.0, 2.5, 5.0 & 10.0 �M) along with optimal level ofauxins NAA (0.5 �M) on in vitro morphogenic response of synthetic seeds was evaluated. The maximumfrequency (86.2%) of conversion of encapsulated beads into plantlets was achieved on MS (M3) mediumcontaining 6-benzyladenine, BA (2.5 �M) and �-naphthalene acetic acid, NAA (0.5 �M) after 4 weeksof culture. Rooting in plantlets was achieved on 1/2 MS + NAA (0.5 �M). Plantlets obtained from storedsynthetic seeds were hardened, acclimatized and established in field, where they grew well without anydetectable malformation. Significant enhancement in the pigment contents (chlorophyll, carotenoidsand net photosynthetic rates) with an increase in acclimatization days may be attributed to chlorophyllbiosynthesis. Activities of antioxidant enzymes i.e. superoxide dismutase, catalase and peroxidase activ-ity) were significantly increased which suggests their preventive role in membrane oxidation and damage

to biological molecules. Also, an enhanced level of lipid peroxidation, as indicated by MDA content, a sen-sitive diagnostic index of oxidative injury clearly indicating its positive determining role in combatingoxidative stress during acclimatization of plantlets. The generated RAPD and ISSR profiles from regen-erated plantlets with mother plant were monomorphic which confirms the genetic stability among the

ed tecange

clones. This synthetic sestorage, germplasm exch

. Introduction

Withania somnifera L. (Dunal) (Solanaceae) commonly referreds Indian ginseng constitute important ingredient in many formu-ations prescribed for a variety of musculoskeletal conditions (e.g.,rthritis, rheumatism), and as a general tonic to increase energy,

mprove overall health and longevity (Chatterjee and Pakrashi,995). Many pharmacological studies have been conducted to

nvestigate the properties of Ashwagandha in an attempt to

Abbreviations: BA, 6-benzyladenine; CaCl2, calcium chloride; EDTA, ethylene-iamine tetraacetic acid; MS, murashige and skoog’s medium; NAA, �-naphthalenecetic acid; NBT, nitroblue tetrazolium; PN, net photosynthetic rate; PVP,olyvinylpyrrollidone; RAPD, random amplified polymorphic DNA; ISSR, inter-imple sequence repeat; SOD, superoxide dismutase; CAT, catalase; POX,eroxidase; MDA, malondialdehyde; TBARS, thiobarbutic acid reactive substances.∗ Corresponding author. Tel.: +91 571 2702016;

E-mail addresses: anismohd37@gmail.com, anism1@rediffmail.com (M. Anis).

926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2013.08.011

hnology could possibly paves the way for the conservation, short-termwith potential storability and limited quarantine restrictions.

© 2013 Elsevier B.V. All rights reserved.

authenticate its use as a multi-purpose medicinal agent. Westernresearch supports its poly-pharmaceutical use, confirming antiox-idant, anti-inflammatory, and anti-stress properties in the wholeplant extract and several separate constituents (Mishra et al., 2000).

A large scale and unrestricted exploitation of the naturalresources to meet its ever-increasing demand by the pharmaceu-tical industry, coupled with limited cultivation and insufficientattempts for its replenishment, have culminated in the markeddepletion of the species. Since, the requirement of W. somniferabiomass rapidly increased over the last few years, concrete meas-ures are needed to conserve this valuable species. Recently, alginateencapsulation technology for the production of synthetic seeds inconjunction with micropropagation has become a viable approachfor in vitro conservation (Ahmad et al., 2012).

Synthetic seed technology is an exciting and rapidly growingresearch area in plant cell and tissue culture (Datta et al., 1999).Production of synthetic seed endowed with high germination rateunder in vitro conditions bears immense potential as an alternative

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f true seeds. Technology offers many useful advantages on a com-ercial scale for the variety of crop plants, especially crops forhich true seeds are not used or readily available for multiplica-

ion or true seeds are expensive, hybrid plants and plants whichre prone to infections. The technology would be useful for multi-lying genetically engineered plantlets (transgenic plants, somaticnd cytoplasmic hybrids, sterile and unstable genotypes).

Plantlets develop within culture vessels on a medium containingmple sugar and nutrient to allow for heterotrophic growth underow levels of light and high relative humidity. These conditionsesult in the formation of plantlets with abnormal morphology,natomy and physiology (Kozai and Smith, 1995). In the course ofardening, the tissue culture plants gradually overcome these inad-quacies and adapt to ex vitro conditions. Plant survival, growth androductivity are reported to be intimately coupled with the aerialnvironment through processes such as energy exchange, loss ofater vapor in transpiration and uptake of CO2 in photosynthe-

is (Stoutjesdijk and Barkman, 1992). Therefore, role of oxidativetress and protective enzymatic systems in relation to progressionf acclimatization process is an essential step to be studied. Theseriteria would be able to supply more objective information thangronomic parameters or visual assessment when evaluating foromponent traits of complex characters.

Genetic stability and maintenance of germplasm is one ofhe most important pre-requisite in the in vitro propagation oflant species. The occurrence of cryptic genetic defects arising viaomaclonal variation in the regenerants can seriously limit theroader utility of the micropropgation system (Salvi et al., 2001).t is therefore imperative to establish genetic uniformity of syn-hetic seed derived plantlets to suggest the quality of the plantletsor its commercial utility. Several strategies have been developedo assess the genetic purity of tissue cultured raised plants such as

orphological descriptions, physiological supervisions, cytologi-al studies, isoenzymes etc., (Gupta et al., 1999). Polymerase chaineaction (PCR) based techniques such as random amplified poly-orphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are

mmensely useful in establishing the genetic stability of in vitro-egenerated plantlets in many plant species (Ahmad and Anis,011; Faisal et al., 2012). RAPD and ISSR markers are very simple,ast, cost-effective, highly discriminative and reliable. They requirenly a small quantity of DNA sample and they do not need anyrior sequence information to design the primer. Since, uniformityf the tissue culture raised progeny is the major concern to maintainhe quality of germplasm, we have adopted RAPD/ISSR techniqueWilliams et al., 1990) for the evaluation of genetic integrity amongegenerated plantlets.

So far, there is only one report available on the developmentf synthetic seed system in W. somnifera using apical buds (Singht al., 2006b). However, no studies available on the possible rolef oxidative stress, protective enzymatic system with their corre-ponding isoenzymes as well as the analysis of genetic fidelity ofynseed derived plantlets of W. somnifera have been reported.

Therefore, the present study has been conducted to optimize thearameters for the production, conservation and their conversionotential under in vitro conditions after cold storage using syntheticeed technology to ensure steady supply of quality plants. More-ver, the physiological, enzymatic activity and the genetic stabilityf the synthetic seed derived plantlets were also assessed duringcclimatization.

. Materials and methods

.1. Encapsulation material

Nodal segments with axillary buds approximately 1 cm dissec-ed aseptically from in vitro established (8 weeks old) cultures of

Products 50 (2013) 468– 477 469

W. somnifera were used as explants for encapsulation (Fatima andAnis, 2012).

2.2. Encapsulation matrix

Different concentrations 2, 3, 4 and 5% (w/v) of sodium algi-nate were prepared using liquid MS medium. For complexation25, 50, 75 100 and 200 mM Calcium chloride solutions were pre-pared. Both, the gel matrix and complexing agent were sterilizedby autoclaving at 121 ◦C (15 lbs) for 15 min after adjusting the pHto 5.8.

2.3. Encapsulation, planting media and culture conditions

Encapsulation was accomplished by mixing the nodal segmentsfrom in vitro regenerated shoots into the sodium alginate solu-tion and dropping them into the calcium chloride solution. Thedroplets containing the explants were held for at least 30 min toachieve polymerization of the sodium alginate. The alginate beadswere then collected, rinsed with sterile liquid MS medium andtransferred to sterile filter paper in petri-dishes for 5 min underthe laminar airflow hood to eliminate the excess of water andthereafter planted into petri-dishes containing sowing medium(M1–M5) composed of MS nutrient medium with the various con-centrations and combinations of BA (0.5, 1.0, 2.5, 5.0 and 10.0 �M)and NAA (0.5 �M), respectively (Fatima and Anis, 2012).

2.4. Low temperature storage

Synthetic seeds (encapsulated nodal segments) weretransferred in petri-dishes containing agar medium M3 (BA2.5 �M + NAA 0.5 �M) and stored in a laboratory refrigerator at4 ◦C. Five different low temperature exposure times (2, 4, 6 and8 weeks) were evaluated for regeneration. After each storageperiod, encapsulated nodal segments were placed on MS mediumwith or without growth regulators for conversion into plantlets.The percentage of encapsulated nodal segments forming shootand root were recorded after 4 weeks of culture to regenerationmedium (M3).

2.5. Hardening and acclimatization

Plantlets with shoot and roots were removed from the cul-ture medium, washed gently with tap water and transferred tothermocol cups containing sterile soilrite, moistened with 1/2 MSlacking organic supplements placed under low light intensity of25 �mol m−2 s−1. Potted plantlets were covered with a transpar-ent polythene membrane to maintain high humidity and irrigatedevery 3 days with half strength MS salt solution lacking vitaminsand PGRs for 2 weeks. The membranes were removed after 2 weeksin order to acclimatize plants to field conditions. After 4 weeks,acclimatized plants were transferred to pots containing normal soil,maintained in a greenhouse and finally transferred to field underfull sun.

2.6. Chlorophyll and carotenoids estimation

The chlorophyll a and b and carotenoids from leaf tissue wereestimated by using the method of McKinney (1941) and Maclachanand Zalik (1963) respectively. About 100 mg fresh tissues frominterveinal areas of leaves were taken after 0 (persistent leaves),7, 14, 21 and 28 (fully expanded leaves) days of acclimatization.

These leaves were grind in 5 ml acetone (80%) with the help ofmortar and pestle. The suspension was filtered with Whatman fil-ter paper number-1, if necessary the supernatant was regrinded,washed and filtered, the total filtrate was taken in graduated test

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ubes and final volume was made upto 10 ml with 80% acetone.he optical density (O.D.) of chlorophyll solution was read at 645nd 663 nm wave lengths and for carotenoids, the O.D. was read at80 and 510 nm wavelengths with the help of a spectrophotometerUV-Pharma Spec 1700, Shimadzu, Japan).

.7. Leaf gas exchange measurements

The net photosynthetic rate (PN) of in vitro regenerated plantsas measured during different stages (0, 7, 14, 21 and 28 days) of

cclimatization using portable Infra Red Gas Analyzer (IRGA, LICOR400, Lincoln, USA) on the basis of net exchange of CO2 between

eaf and atmosphere by enclosing the leaf in the leaf chamber, andonitoring the rate at which the CO2 concentration changed over

short time intervals (10–20 s). The net photosynthetic rate wasxpressed as �mol CO2 m−2 s−1.

.8. Antioxidant enzymes extraction and assay

To determine antioxidant enzyme levels, 0.5 g fresh leaf tis-ue was homogenized in 2.0 ml extraction buffer containing 1%olyvinylpyrrollidone (PVP), 1% Triton x-100, and 0.11 g ethylene-iamine tetraacetic acid (EDTA) using pre-chilled mortar andestle. The homogenate was filtered through four layers of cheeseloth and centrifuged at 15,000 rpm for 20 min. The supernatantas used for enzyme assays. Extraction was carried out in dark at◦C.

.9. Superoxide dismutase (SOD) estimation

Superoxide dismutase (superoxide:superoxide oxireductase, EC.15.1.1) activity was measured by method of Dhindsa et al. (1981)ith slight modifications. SOD activity in the supernatant was

ssayed by its ability to inhibit the photochemical reduction. Theeaction mixture consisting of 0.5 M potassium phosphate buffer,00 mM methione, 1 M sodium carbonate, 2.5 mM nitroblue tetra-olium (NBT) solution, 3 mM EDTA, 0.1 ml enzyme extract, 60 �Miboflavin, and 1.0 ml DDW were incubated in a test tube under

15 W fluorescent lamp (Phillips, India) for 10 min at 25–28 ◦C.eaction mixture containing all the above substances along withnzyme, placed in dark served as blank A whereas blank B con-ained all the above substances except enzyme and placed in lightlong with the sample. Absorbance of samples along with blank Bas read at 560 nm against the blank A. A 50% reduction in color was

onsidered as one unit of enzyme activity. SOD activity of extractas expressed as enzyme units (EU) mg−1 protein min−1.

.10. Estimation of catalase (CAT) activity

Catalase (H2O2:H2O2 oxidoreductase: EC 1.11.1.6) activity in theeaves of regenerated plantlets was determined by the method ofebi (1984) with slight modifications. Reaction mixture contain-

ng 0.5 M potassium phosphate buffer, 3 mM EDTA, 0.1 ml enzymextract, and 3 mM H2O2. The reaction was allowed to run for 5 min.atalase activity was determined by monitoring the disappearancef H2O2 and measuring the decrease in absorbance at 240 mm.AT activity was calculated by using extinction coefficient (D ).036 mM−1 cm−1 and expressed in enzyme units (EU) mg−1 pro-ein min −1. One unit of enzyme determines the amount necessaryo decompose 1 �mol of H2O2 per min at 25 ◦C.

.11. Estimation of peroxidase

Peroxidase content (EC 1.11.1.7) was determined by Bergmeyert al. (1974). About 0.2 mg fresh leaves sample were collected andomogenized in a mortar and pestle with 5 ml chilled phosphate

Products 50 (2013) 468– 477

buffer (50 mM pH 7.8). The homogenate centrifuged 10, 000 rpmfor 20 min at 4 ◦C. The supernatant was stored at 4 ◦C and used forthe peroxidase assay. The assay mixture contains 0.1 M phosphatebuffer (pH 7.8), 4 mM pyrogallol, 3 mM H2O2 and crude enzymeextract. Transfer the reaction mixture into a suitable cuvette andmeasure the absorbance at 420 nm using Spectrophometer (UV-Pharma Spec 1700, Shimadzu, Japan). The enzyme was expressedas �mole (H2O2 destroyed) mg−1 (protein) s−1. The peroxidasecontent was determined as follows:

Unit/g material = (A 420 nm/ min) (Df ) (1000) (Vt)(ε) (Vs) (Cc)

wherein, Df is the Dilution factor (21); 1000 is the conversion factorfrom mg to g; Vtis the final volume of reaction mixture; ε is the mil-limolar extinction coefficient of purpurogallin at 420 nm; Vs is thevolume of enzyme used; Cc is the enzyme concentration in mg/ml.

It measures the oxidation of pyrogallol to purpurogallin by per-oxidase when catalyzed by peroxidase at 420 nm.

H2O2 + pyrogallol

(Donor)

peroxidase−→2H2O + purpuogallin

(Oxidized donor)

2.12. Estimation of carbonic anhydrase (CA) activity

The enzyme CA catalyses the reversible hydration of carbondioxide (CO2) to give the bicarbonate ion. It was assayed using themethod of Dwivedi and Randhawa (1974).

H2O + CO2carbonic anhydrase−→ H+ + HCO−

Plant leaves were sampled from each sample randomly. Leaveswere cut into small segments (1 cm2) at a temperature below 25 ◦C.After mixing them, 200 mg leaf pieces were weighed and cut fur-ther into small pieces (2–3 mm length) in 10 ml 0.2 M cystein in apetridish at 0–4 ◦C. After being cut, the solution adhering at theirsurface was removed with the help of a blotting paper followed bytransfer immediately to a test tube, having 4 ml phosphate bufferof pH 6.8. To this 3.4 ml 0.2 M sodium bicarbonate (NaHCO3) in0.02 M sodium hydroxide (NaOH) solution and 0.2 ml 0.002% bro-mothymol blue indicator was added. After shaking, the tube waskept at 0–4 ◦C for 20 min.

2.13. Estimation of lipid peroxidation

Thiobarbutic acid reactive substances (TBARS) content wasdetermined using a modified protocol of Cakmak and Horst (1991).The lipid peroxidation product in leaf samples was expressed asMDA (malondialdehyde) content. Approximately 0.5 g leaf tissuewas homogenized with 5 ml 0.1% trichloroacetic acid (TCA), andcentrifuged at 15,000 rpm for 5 min. Then, 1 ml aliquote of thesupernatant was mixed with 4 ml 0.5% (w/v) thiobarbituric acid(TBA), prepared in 20% (w/v) TCA, and incubated in boiling waterfor 30 min. Thereafter, it was immediately cooled on ice to stop thereaction, and centrifuged at 12,000 rpm for 30 min. The supernatantwas placed in a UV-VIS spectrophotometer (UV-1700 PharmaSpec)to determine the absorbance at 532 nm and corrected for non-specific turbidity by subtracting its absorbance at 600 nm. Theconcentration of lipid peroxides together with oxidatively modi-fied proteins of plants were thus quantified in terms of MDA levelusing an extinction coefficient of 155 mM−1 cm−1 and expressed as

nmol g−1 fresh weight. TBARS content was determined as follows.

TBARS content(nmole g−1 fresh weight)

= (A532 − A600)V × 1000/ε × W

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herein, � is the specific extinction coefficient (155 mM−1 cm−1); is the volume of the extraction medium; W is the fresh weightf leaf; A600 is an absorbance at 600 nm; A532 is an absorbance at32 nm.

.14. Evaluation of genetic stability of the micropropagatedecovered plants

The genetic stability of the recovered plants was studied byAPD and ISSR techniques. Ten micropropagated plants from fieldransferred progeny were randomly selected alongwith motherlant and evaluated for genetic homogeneity. Genomic DNA wasxtracted from young leaves of W. somnifera, following the cetyl-ethylammonium bromide (CTAB method) described by Doyle andoyle (1990). The extracted DNA was tested for purity (A260/280

atio) on a UV–vis spectrophotometer (UV-1700 Pharma Spec, Shi-adzu, Japan, Kyoto).A set of thirteen ISSR (UBC, Vancouver, BC, Canada) and 40

APD (Kit OPB and OPC) primers were used for initial screening.CR reactions for RAPD/ISSR markers amplification were performedn a thermocycler (Biometra, T Gradient Thermoblock, Germany).he PCR amplification mixture (20 �l) contained 10X buffer (2 �l),

ig. 1. Plant regeneration from encapsulated nodal segments of W. somnifera. (A) Artifiodium alginate and 100 mM calcium chloride. (B) Culture showing shoot emergence fromn MS + BA (2.5 �M) + NAA (0.5 �M) after 4 weeks of culture. (D) Acclimatized plantlets d

Products 50 (2013) 468– 477 471

25 mM MgCl2 (1.2 �l), 10 mM dNTPs (0.4 �l), 2 �M primers, 3 UnitTaq polymerase (0.2 �l) and 40 ng Template DNA. PCR amplifica-tion program consisted of 45 cycles including a 94 ◦C denaturationstep of 5 min, a 35 ◦C annealing for 1 min and a 72 ◦C elongationof 1 min. A final extension was followed at 72 ◦C for 10 min. DNAamplification products were fractioned by electrophoresis in 0.8%(w/v) agrose gels with 4 �l ethidium bromide in TAE buffer (pH 8.0)run at 50 V for 2 h and visualized on a UV transilluminator (Bio Rad,Hercules, CA, USA). In order to assess the consistency of band pro-files DNA isolation and PCR reactions were carried out three times.Only well defined and reproducible bands were scored. Bands withthe same migration were considered to be homologous fragments,regardless of intensity.

2.15. Statistical analysis

All the experiments were repeated thrice with 20 nodal seg-ments for each treatment. The data obtained were analyzed using

statistical software, SPSS Version 16 (SPSS Inc. Chicago, USA) andmeans were compared using Duncan’s multiple range test (DMRT)at 0.5% level of significance. All the results were expressed inmean ± standard error.

cial seeds of W. somnifera obtained by the encapsulation of nodal segments in 3% synthetic seeds after 2 weeks of culture. (C) Germinated synthetic seed with shooterived from encapsulated nodal segments.

472 N. Fatima et al. / Industrial Crops and Products 50 (2013) 468– 477

Table 1Effect of different concentrations of sodium alginate with optimum concentra-tion of calcium chloride (100 mM) on the formation and conversion of syntheticseeds of W. somnifera after 4 weeks of culture on MS medium. Values representmeans ± standard error of 20 replicates per treatment in three repeated experi-ments. Means followed by the same letter are not significantly different (P = 0.05)using Duncan’s multiple range test.

Sodium alginate(% w/v)

Conversion response Remarks

1 0.00 ± 0.00d Fragile beads difficult to handle2 0.00 ± 0.00d -do-3 92.4 ± 0.64a Beads with soft texture

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also served as an artificial endosperm, thereby providing nutrientsto the encapsulated explants for regrowth. Antonietta et al. (1999)reported that the synthetic endosperm should contain nutrientsand a carbon source for germination and conversion.

n

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4 76.6 ± 0.90b Hard beads5 58.1 ± 0.49c Hard beads

. Results and discussion

The encapsulated beads differed morphologically with respecto texture, shape and transparency with different combinationnd concentrations of sodium alginate (2–5%) and CaCl2. 2H2O225–200 mM). The assessment of the effects of various concentra-ions of sodium alginate and calcium chloride was prerequisite inrder to standardize the preparation of characteristic beads. Anncapsulated matrix of 3% sodium alginate with 100 mM CaCl2.H2O2 was found most suitable for the formation of clear, uni-orm beads within ion exchange duration of 30 min (Fig. 1A;ables 1 and 2). Higher concentration of sodium alginate (4% or%) and calcium chloride (200 mM) were found to be unsuitableecause the resulting beads formed were too hard, isodiametricnd also inhibits the conversion rate while, the lower concentra-ion of sodium alginate (1 or 2%) and CaCl2 (25 or 50 mM) notnly prolonged the ion exchange (polymerization) duration butlso resulted in the formation of fragile beads that were difficulto handle (Tables 1 and 2). Sodium alginate preparations at loweroncentration were unsuitable, probably because of a reduction inheir gelling ability after exposure to high temperature during auto-laving (Larkin et al., 1988). A successful propagation system routedhrough encapsulation is based on significant evaluation of factorsffecting gel matrix and also on sodium alginate and CaCl2. 2H2O,hich plays an important role in complexion and capsule quality

Singh et al., 2006a). The present results are in corroboration withrevious findings (Siddique and Anis, 2009).

Nodal segments encapsulated in 3% (w/v) sodium alginate and00 mM CaCl2. 2H2O exhibited shoot regrowth after 2–3 weeksFig. 1B), cultured on five different media (M1–M5), describedbove. The frequency of shoot development varied with mediumomposition. The highest frequency (86.2%) of conversion of encap-ulated buds was achieved on MS medium (M3) supplementedith BA (2.5 �M) and NAA (0.5 �M) after 4 weeks of culture

Figs. 1C and 2). Conversion into plantlets was achieved after 6

eeks of culture on the same medium. Shoots developed werehenotypically normal with distinct nodes and internodes. Thereas no regeneration occurred on hormone free medium (M1)

able 2ffect of different concentrations of calcium chloride with optimum level of sodiumlginate (3%) on the formation and conversion of synthetic seeds of W. somniferafter 4 weeks of culture on MS medium. Values represent means ± standard error of0 replicates per treatment in three repeated experiments. Means followed by theame letter are not significantly different (P = 0.05) using Duncan’s multiple rangeest.

Calcium chloride (mM) Conversion response Remarks

25 0.00 ± 0.00d Fragile beads difficult to handle50 0.00 ± 0.00d -do-75 84.4 ± 1.44b Very soft but handled easily

100 97.0 ± 0.51a Soft beads and easy to handle200 69.4 ± 0.20c Hard beads

represent means ± standard error of 20 replicates per treatment in three repeatedexperiments. Means followed by the same letter are not significantly different(P = 0.05) using Duncan’s multiple range test.

whereas, higher concentration of hormone (M5) in a mediumshowed the emergence of weak shoots with stunted growth. This isin accordance with the previous findings (Kavyashree et al., 2006).

Storage duration (2, 4, 6 and 8 weeks) was also found to influencethe regeneration frequency of encapsulated axillary buds at 4 ◦C. Ahighly desirable feature of encapsulated nodal segment is their abil-ity to retain viability in terms of sprouting and conversion potentialeven after a considerable period of storage required for germplasmexchange. The effect of different storage duration on encapsulatednodal segment at 4 ◦C is summarized in Fig. 3. An alginate matrix

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Fig. 3. Effect of cold storage (4 ◦C) on in vitro regeneration from alginateencapsulated nodal cuttings of W. somnifera on M3 medium. Values representmeans ± standard error of 20 replicates per treatment in three repeated experi-ments. Means followed by the same letter are not significantly different (P = 0.05)using Duncan’s multiple range test.

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acclimatization (Fig. 7). SOD is believed to play a crucial role inthe antioxidant systems as it catalyses the dismutation of O2 intoH2O2 and O2 (Bowler and Van Montagu Inze, 1992). In response

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N. Fatima et al. / Industrial Cro

A decline in percentage conversion frequency (62.8%) of syn-hetic seed was observed as the storage period increased beyond 4eeks. This decline in the conversion response could be attributed

o the inhibition of tissue respiration by the alginate matrixRedenbaugh et al., 1984) or a loss of moisture due to the partialesiccation during storage (Danso and Ford-Lloyd, 2003). Encap-ulated nodal segments were viable up to (50.5%), even after 8eeks of cold-dark storage (Fig. 3). These observations corrobo-

ates with earlier findings by Faisal and Anis (2007). The presenttudy could be considered as an improved encapsulation techniqueor W. somnifera using nodal explants. In the present investiga-ion, the conversion frequency of synseed was quite higher (86.2%)fter 4 weeks of cold storage in comparison to other reports on W.omnifera (Singh et al., 2006b).

The synthetic seeds demonstrated high adventitious rootingapacity after sowing. The regenerated micro-shoots rooted whenxcised and subjected to 1/2 MS medium containing NAA (0.5 �M).ooted plantlets with 4–5 fully developed leaves, retrieved fromncapsulated nodal segments were transferred to thermocol cupsontaining soilrite. The plantlets were covered with transparentolybags and acclimatized by adopting the standard procedure.fter 4 months, they were transferred to pots containing normalarden soil and maintained in greenhouse with 90% survival rateFig. 1D). Micropropagation is restricted by often high percentage oflants lost or damaged during ex vitro transplantation (Pospisilovat al., 1999). The plantlets are susceptible to various stresses. Awitch to autotrophy and changes in stomata functioning anduticle compositions has been reported during acclimatizationHuylenbroeck et al., 1998). The imposition of environmentaltresses increased the rate of production of reactive oxygen speciesROS). ROS are inevitable byproducts of aerobic metabolism whichause lipid peroxidation and consequently membrane injuries, pro-ein degradation, enzyme inactivation, damage to DNA etc. Toounter the hazardous effects of reactive oxygen species, plant cellsevelop a complex antioxidant defense and enzymatic scavengingystem composed of antioxidant enzymes and metabolites suchs superoxide dismutase (SOD), catalase (CAT) and peroxidasesPOXs) etc.

During transfer of tissue cultured raised plantlets from in vitroo ex vitro, the change in pigment concentration (Chlorophyll andarotenoid contents) was estimated and it was observed thatith an increase in number of days of acclimatization, the pig-ent contents increased significantly. The Chl a content was low

0.23 ± 0.01/) mg g−1 on 0 days of acclimatization whereas, it wasncreased upto (0.42 ± 0.02) mg g−1 after 21 days and was maxi-

um (0.45 ± 0.01) mg g−1 at 28 days of transfer (Fig. 4). Decreasedn chlorophyll level during the first week of transplantation wasccompanied by poorly developed chloroplast and disorganizedrana. Enhancement in pigment contents may be attributed to thenduction of chlorophyll synthesis enzyme required for chlorophylliosynthesis. Similar results have been reported by Pospisilova et al.1999) and Jeon et al. (2005).

Carotenoid plays an important role in protection of chlorophylligments under stress conditions (Kenneth et al., 2000) whichight be generated during acclimatization. Carotenoid contents

ncreased gradually during the period of transplantation. The max-mum carotenoid level (0.10 ± 0.01) mg g−1 was observed after 28ays of acclimatization (Fig. 4). Increase in carotenoid levels is notnexpected as carotenoids are reported to be involved in protectinghe photosynthetic machinery from photo-oxidative damage (Jeont al., 2005).

During transplantation of plantlets, the change in net photo-

ynthetic rate (PN) was observed after 7, 14, 21 and 28 days ofcclimatization. PN as measured decreased in the first week afterransplantation and increased thereafter. The highest net photo-ynthetic rate (4.10 ± 0.14) �mol CO2 m−2 s−1 was obtained after

by the same letter are not significantly different (P = 0.05) using Duncan’s multiplerange test.

28 days of transplantation to ex vitro environment (Fig. 5). Thedecline in photosynthetic rate during the first week after thetransfer from in vitro to ex vitro condition indicates that climaticconditions create stress in micropropagated plants. Similar resultswere observed earlier in Rosa hybrida (Genoud-Gourichon et al.,1999).

Carbonic anhydrase activity was assessed during acclimatiza-tion process and was found to increase during hardening processand reached (1.90 ± 0.10) mM CO2 g−1 fresh mass−1 after threeweeks of transfer (Fig. 6). The enzymes play a determinant rolein transport and exportation of sugar with the plant (Aragon et al.,2005).

Acclimatized plantlets of W. somnifera showed a time dependentincrease in superoxide dismutase, catalase and peroxidase activity.SOD, POX and CAT are the key enzymes involved in the detoxifi-cation of the deleterious oxygen species. Changes in SOD activitywas observed during the first 14 days after transplantation andreached maximum (9.20 ± 0.10) mg−1 protein min−1 at 28 days of

Fig. 5. Changes net photosynthetic rate (�mol CO2 m−2 s−1) during acclimatizationof W. somnifera plantlets. Values represent means ± standard error of 20 replicatesper treatment in three repeated experiments. Means followed by the same letterare not significantly different (P = 0.05) using Duncan’s multiple range test.

474 N. Fatima et al. / Industrial Crops and Products 50 (2013) 468– 477C

A a

cti

vit

y [

mM

CO

2 (

g-1

) fr

es

h m

as

s s

-1]

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

CA activity

0 7 14 21 28

Acclimat izati on period (da ys)

e

d

b

ca

Fig. 6. Changes in carbonic anhydrase activity [mM CO2 (g−1) fresh mass s−1] duringacclimatization of W. somnifera plantlets. Values represent means ± standard errorost

t(b(tmt(aieehsHt

oAt

Fopa

Acclimatization perio d ( days)

Cata

las

e a

cti

vit

y (

mo

l m

in-1

mg

-1 p

rote

in)

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Pero

xid

ase a

cti

vit

y [

mo

le m

g-1

(p

rote

in)

s-1

]

0

1

2

3

4

5

CAT act ivity

POX activity

0 7 14 21 28

de

d

c

b

a

d

d

c

b

a

Fig. 8. Changes in CAT (�mol min−1 mg−1 protein) and POX [�mole mg−1 (pro-tein) s−1] activity during acclimatization of W. somnifera plantlets. Values represent

f 20 replicates per treatment in three repeated experiments. Means followed by theame letter are not significantly different (P = 0.05) using Duncan’s multiple rangeest.

o this, the cellular machinery generates free radical scavengersSOD) that effectively prevent membrane oxidation and damage toiological molecules. In addition, peroxidase (POXs) and catalaseCAT) activity increased during the whole period of acclimatiza-ion and reached to its maximum value (4.40 ± 0.10; 4.20 ± 0.10)

g−1 protein min−1 respectively, at 28 days of transfer as comparedo control plantlets (Fig. 8). The increase in antioxidants activitiesSOD, POX and CAT) was noticed upto 28 days of acclimatizationnd it got stabilized thereafter. The augmentation in catalase activ-ty could be explained by peroxisomal proliferation, where thisnzyme is localized (Willekens et al., 1995). Increased catalase lev-ls also suggest its role in the photo-respiratory detoxification ofydrogen peroxide through the mitochondrial electron transportystem (Scandalios, 1990). POXs catalyses various reactions where2O2 is used as one of their substrates including cell wall lignifica-

ions (Lee et al., 2007).

The ROS-induced peroxidation of lipid membrane is a reflection

f stress-induced damage at the cellular level (Jain et al., 2001).n enhanced level of lipid peroxidation, as indicated by MDA con-

ent, was observed in Withania leaves after transplantation to ex

SO

D a

cti

vit

y (

Un

it m

g-1

pro

tein

min

-1 )

4

5

6

7

8

9

10

SOD activity

de

c

b

a

0 7 14 21 28

Acclimatization period (da ys)

ig. 7. Changes in SOD (Unit mg−1 protein min−1) activity during acclimatizationf W. somnifera plantlets. Values represent means ± standard error of 20 replicateser treatment in three repeated experiments. Means followed by the same letterre not significantly different (P = 0.05) using Duncan’s multiple range test.

means ± standard error of 20 replicates per treatment in three repeated experi-ments. Means followed by the same letter are not significantly different (P = 0.05)using Duncan’s multiple range test.

vitro environment, clearly indicating an oxidative stress. MDA is acommon product of lipid peroxidation and a sensitive diagnosticindex of oxidative injury (Janero, 1990). During transplantation,the MDA contents in leaves was found to be low (2.53 ± 0.13)�mole mg−1 s−1 after 7 days but the MDA level significantlyincreased to (3.86 ± 0.06) �mole g−1 FW after 28 days of acclimati-zation (Fig. 9). The increase in TBARs (Thiobarbituric acid reactivesubstances) content observed in this study may be an indicator ofthe regeneration of ROS. Similar findings are in accordance withFaisal and Anis (2009). Increase in lipid peroxidation was reportedin many plants under various environment stresses (Prasad, 1996).

RAPD and ISSR finger printing of ten randomly selected, in vitroraised plants and mother plant was carried out. Well resolved, clearand distinct banding patterns were manually scored from the gelprofiles and included for final analysis. Bands with same mobilitywere treated as identical fragments and weak bands were excludedfrom the final analysis. RAPD and ISSR were chosen because of their

simplicity and cost-effectiveness. They amplify different regions ofthe genome providing broad analysis of genetic stability or vari-ation in plants. Out of 20 RAPD primers screened from each Kit B

MD

A C

on

ten

t (

mo

l g

-1 F

W)

0

1

2

3

4

5

MDA conte nts

a

a

b

b

c

0 7 14 21 28

Acclimatiza tion per iods (da ys)

Fig. 9. Changes in MDA (�mol g−1 FW) concentration during acclimatization of W.somnifera plantlets. Values represent means ± standard error of 20 replicates pertreatment in three repeated experiments. Means followed by the same letter arenot significantly different (P = 0.05) using Duncan’s multiple range test.

N. Fatima et al. / Industrial Crops and Products 50 (2013) 468– 477 475

Table 3Randomly amplified polymorphic DNA primers (RAPD) used to screen ten micropropagated plantlets.

S. No. Kit B Kit C

Primers Sequence (5′-3′) No. of bands Primers Sequence (5′-3′) No. of bands

1 OPB01 GTTTCGCTCG 2 OPC01 TTCGAGCCAG 42 OPB02 TGATCCCTGG 4 OPC02 GTGAGGCGTC 23 OPB03 CATCCCCCTG 7 OPC03 GGGGGTCTTT 54 OPB04 GGACTGGAGT 9 OPC04 CCGCATCTAC 35 OPB05 TGCGCCCTTC 3 OPC05 GATGACCGCC 36 OPB06 TGCTCTGCCC 3 OPC06 GAACGGACTC 77 OPB07 GGTGACGCAG 2 OPC07 GTCCCGACGA Nil8 OPB08 GTCCACACGG Nil OPC08 TGGACCGGTG 119 OPB09 TGGGGGACTC Nil OPC09 CTCACCGTCC 5

10 OPB10 CTGCTGGGAC 5 OPC10 TGTCTGGGTG 411 OPB11 GTAGACCCGT 4 OPC11 AAAGCTGCGG 412 OPB12 CCTTGACGCA 6 OPC12 TGTCATCCCC 313 OPB13 TTCCCCCGCT 4 OPC13 AAGCCTCGTC 214 OPB14 TCCGCTCTGG 5 OPC14 TGCGTGCTTG 415 OPB15 GGAGGGTGTT Nil OPC15 GACGGATCAG 616 OPB16 TTTGCCCGGA 5 OPC16 CACACTCCAG 317 OPB17 AGGGAACGAG 6 OPC17 TTCCCCCCAG 2

awoubb

er8

Flmaa

duced monomorphic pattern across all the plants and the motherplant, confirming the genetic uniformity of the micropropagatedplantlets. Our results corroborate with the earlier reports on genetic

18 OPB18 CCACAGCAGT Nil

19 OPB19 ACCCCCGAAG 3

20 OPB20 GGACCCTTAC 2

nd Kit C, 16 and 18 primers produced clear, reproducible bands andell resolved banding pattern, respectively (Table 3). On the basis

f banding pattern and resolution, primers OPB 04 and OPC 08 weresed for further analysis. Primer OPB 04 produced 9 monomorphicands (Fig. 10A), while primer OPC 08 produced 11 monomorphicands (Fig. 10B; Table 3).

For ISSR analysis, 13 ISSR primers were screened for the regen-

rated plants. All the 13 primers gave clear, unambiguous andeproducible bands and were used for ISSR-PCR. Primer UBC-66 amplified maximum 13 monomorphic bands (Fig. 11A), while

ig. 10. A profile of polymerase chain reaction (PCR) amplification products fromane 1–10 micropropagated plants of W. somnifera using Randomly amplified poly-

orphic DNA (RAPD) primer OPB 04. A profile of polymerase chain reaction (PCR)mplification products from lane 1–10 micropropagated plants using randomlymplified polymorphic DNA (RAPD) primer OPC 08.

OPC18 TGAGTGGGTG 5OPC19 GTTGCCAGCC NilOPC20 ACTTCGCCC 2

primer UBC 891 produced 9 monomorphic bands (Fig. 11B; Table 4).No polymorphism was detected during the ISSR analysis of tissueculture raised plantlets. All the RAPD and ISSR tested primers pro-

Fig. 11. A profile of polymerase chain reaction (PCR) amplification products fromlane 1–10 micropropagated plants using inter sequence repeat (ISSR) primer UBC866. A profile of polymerase chain reaction (PCR) amplification products from lane1–10 micropropagated plants using Inter sequence repeat (ISSR) primer UBC 891. M,Marker (�DNA/EcoR1 + HindIII indicated in bp); P, Donor plant; Lane 1–10, Micro-propagated plants.

476 N. Fatima et al. / Industrial Crops and

Table 4Inter sequence repeats (ISSR) primers were used to verify the genetic fidelity ofmicropropagated plantlets.

S. No. Name of primers Primers sequences (5′-3′) No. of bands

1 UBC-801 (AT)8T 32 UBC-811 (GA)8C 73 UBC-825 (AC)8T 44 UBC-827 (AC)8G 65 UBC-834 (AG)8YT 56 UBC-841 (GA)8YC 57 UBC-855 (AC)8YT 48 UBC-866 (CTC)6 139 UBC-868 (GAA)6 8

10 UBC-880 (GGGGT)3G 7

s(

4

qswetseithdaioscittg

A

F(R((

R

AA

A

A

A

11 UBC-889 DBDA(CA)6C 512 UBC-891 HVHT (GT)6G 913 UBC-900 ACTTCCCCACAGGTTAACAC 6

tability of synthetic seed derived plantlets of Cineraria maritimeSrivastava et al., 2009) and Rauvolfia serpentina (Faisal et al., 2012).

. Conclusion

The present protocol highlights the development of high fre-uency shoot recovery in W. somnifera from encapsulated nodalegments after 4 weeks of storage. The technique offers a simpleay of handling cells and tissues, protecting them against strong

xternal gradients and proves an efficient delivery system. Syn-hetic seeds are also expected to offer an appropriate recipientystem for alien gene transfer in micro-projectile based gene deliv-ry system. Since, antioxidant metabolism has been shown to bemportant in determining the ability of plants to survive in oxida-ive stresses, therefore, an up regulation of these enzymes wouldelp to reduce the buildup of ROS. This factor could be a key toesign adequate methods to improve acclimatization process. Thebility to generate transgenic plants provides a powerful tool toncrease the level of stress tolerance by increasing the expressionf the native genes of the antioxidant enzymes, the natural defenseystem in plants. The basic information on different biochemicalhanges during acclimatization process of in vitro raised plantletss also essential for further molecular breeding. The present pro-ocol explores the possibility of preserving the genetic stability ofhe selections and promotes true to type genotype for exchange ofermplasm between laboratories.

cknowledgements

Financial assistance in the form of a Dr. D. S. Kothari Postdoctoralellowship to Nigar Fatima from University Grants CommissionUGC), Government of India, New Delhi, is gratefully acknowledged.esearch support from The Department of Science and TechnologyGovt. of India) New Delhi under the DST- FIST (2011) and UGC-SAP2009) Program, is also acknowledged.

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