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Applied Biochemistry andBiotechnologyPart A: Enzyme Engineering andBiotechnology ISSN 0273-2289Volume 170Number 5 Appl Biochem Biotechnol (2013)170:1163-1173DOI 10.1007/s12010-013-0266-3

An Improved Micropropagation of Arnebiahispidissima (Lehm.) DC. and Assessmentof Genetic Fidelity of MicropropagatedPlants Using DNA-Based MolecularMarkersMahendra Phulwaria, Manoj K. Rai &N. S. Shekhawat

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An Improved Micropropagation of Arnebia hispidissima(Lehm.) DC. and Assessment of Genetic Fidelityof Micropropagated Plants Using DNA-BasedMolecular Markers

Mahendra Phulwaria & Manoj K. Rai & N. S. Shekhawat

Received: 4 November 2012 /Accepted: 24 April 2013 /Published online: 5 May 2013# Springer Science+Business Media New York 2013

Abstract An efficient and improved in vitro propagation method has been developed forArnebia hispidissima, a medicinally and pharmaceutically important plant species of aridand semiarid regions. Nodal segments (3–4 cm) with two to three nodes obtained from fieldgrown plants were used as explants for shoot proliferation. Murashige and Skoog’s (MS)medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) ornaphthalene acetic acid was used for shoot multiplication. Out of different PGRs combina-tions, MS medium containing 0.5 mg l−1 6-benzylaminopurine and 0.1 mg l−1 IAA wasoptimal for shoot multiplication. On this medium, explants produced the highest number ofshoots (47.50±0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under thegreenhouse condition when the base (2–4 mm) of shoots was treated with 300 mg l−1 ofindole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhousewith 85–90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simplesequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened,15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR)reproducible amplicons. The amplified products were monomorphic across all themicropropagated plants and were similar to the mother plant. To the best of our knowledge,it is the first report on the assessment of the genetic fidelity in micropropagated plants ofA. hispidissima.

Keywords Ex vitro rooting . Genetic stability .Molecular markers .Multiplication . Shikonin

Introduction

In recent years, a lot of attention has been devoted to secondary metabolites, also known as“natural products” synthesized by various plants that exhibit a range of clinical and

Appl Biochem Biotechnol (2013) 170:1163–1173DOI 10.1007/s12010-013-0266-3

M. Phulwaria (*) :M. K. Rai :N. S. ShekhawatBiotechnology Unit, Department of Botany, Jai Narain Vyas University, Jodhpur 342001, Rajasthan, Indiae-mail: [email protected]

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pharmacological activities. Secondary metabolites are the products of metabolism and are notessential for normal growth, development, or reproduction of an organism. However, they playan important role in determining the quality of food and widely used as drugs, dyes, flavors,fragrance, and insecticides [1, 2]. Recently, Vaishnav and Demain [2] reviewed on the alterna-tive applications of secondary metabolites which include antitumor, cholesterol lowering,immunosuppressant, antiprotozoal, antihelminthic, antiviral, and antiaging activities. Due tothe increasing human populations and excess demand of herbal medicine and secondarymetabolites, the status of many commercially important plants has now been affected in naturalpopulation. During the last three to four decades, plant tissue culture is used as a valuable toolfor the conservation and rapid propagation of medicinally important plants and the productionof secondary metabolites [3–6]. Shikonin/alkannin was the first secondary metabolite producedon commercial level using biotechnological methods [7]. Through tissue culture techniques,specific secondary metabolites can be produced in cell, tissue, and organ culture throughout theyear even at the place where these particular plants are not grown [8]. However, under theinfluence of tissue culture conditions, there are chances of getting variations in in vitro-regenerated plants. Therefore, the maintenance of genetic fidelity of tissue culture-raised plantswith respect to the mother plant is an essential requisite for upholding certain traits, particularlythe nature and content of secondary metabolites in case of medicinal plants [9]. In recent years,several DNA-based molecular markers are used for assessment of genetic stability of in vitro-regenerated plantlets. Among them, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers are the simple, fast, and cost-effective methods [10,11]. These twomarkers have been successfully used to analyze genetic stability among in vitro-regenerated plantlets of many plant species [10–17].

Arnebia hispidissima (Lehm.) DC., which belongs to the family Boraginaceae, is amedicinally and pharmaceutically important plant widely distributed in semiarid regionsof Rajasthan. Fresh flowers of A. hispidissima produced a number of flavonoids namelyapigenin, cyanidin, kaempferol, luteolin, and quercetin [18]. The alkannin and its opticalisomer shikonin, a red dye and root-specific secondary metabolites of A. hispidissima, haveanti-inflammatory, antimicrobial, and antitumor activities and wound healing properties. Inaddition, alkannin is well known for its applications in the treatment of ulcers, boils, cuts,heart ailments, headache, fever, and tongue and throat troubles and used as a colorant infood, cosmetic, and textiles industries [4, 18–21]. Earlier, some reports are available whichdocument in vitro plant regeneration of this important medicinal plant [4, 21]. However,main emphasis of these studies was the alkannin production from callus or cell suspensionculture. More recently, we reported shoot regeneration of this plant through callus derivedfrom immature inflorescence explants [22].

The present communication describes an improved and efficient protocol for large-scalepropagation of A. hispidissima using nodal segments as explant. The multiplication rateachieved in the present investigation is higher than the earlier reports on the same plant [4,21, 22]. Efforts were also made first time to assess genetic integrity among regenerants anddonor plants using DNA-based molecular markers, i.e., RAPD and ISSR.

Material and Methods

Explant Selection and Surface Sterilization

Fresh shoots/ sprouts from field-growing plants were collected in the months of January–February to establish the cultures. Nodal segments (3–4 cm) with two to three nodes were

1164 Appl Biochem Biotechnol (2013) 170:1163–1173


cut from sprouts and washed thoroughly with tap water for 20–30 min. Explants were treatedinitially with 0.1 % (w/v) Bavistin (BASF India Limited, Mumbai, India; a systemicfungicide) for 8–10 min. Surface sterilization of explants was carried out with 0.1 % (w/v)mercuric chloride (HgCl2) (Hi-Media, India) for 3 min under aseptic condition, and thesurface-sterilized explants were washed seven to eight times with sterile distilled water.

Bud Breaking and Multiple Shoot Induction

The surface-sterilized explants were inoculated on Murashige and Skoog’s (MS) [23]medium with various concentrations (0.0–5.0 mg l−1) of 6-benzylaminopurine (BAP) orkinetin (Kin) for shoot bud induction. MS medium with sucrose (3 %) and additives(50 mg l−1 of ascorbic acid and 25 mg l−1 each of adenine sulphate, arginine, and citricacid) were used throughout the experiment. The pH of the medium was adjusted to 5.8±0.02and autoclaved for 15 min at 121 °C. The cultures were maintained at 28±2 °C under a 14-hphotoperiod with a light intensity of 40–50 μmol m−2 s−1 photon flux density provided bycool white fluorescent lamps (Philips, India) and 60 % relative humidity (RH). For shootmultiplication, the in vitro-regenerated shoots were cut into segments (1.0–2.0 cm in length)each with one to two nodes and subcultured on MS medium containing various concentra-tions of BAP (0.0–1.5 mg l−1) alone or optimized concentration of BAP (0.5 mg l−1) withvarious concentrations (0.0–0.3 mg l−1) of indole-3-acetic acid (IAA) or naphthalene aceticacid (NAA).

Ex Vitro Rooting and Hardening of Cloned Plantlets

For root induction under ex vitro condition, in vitro-regenerated shoots (3–5 cm) wereexcised from shoot clumps and treated with different concentrations (0.0–500 mg l−1) ofNAA or indole-3-butyric acid (IBA) for 5 min. The auxin-treated shoots were transferred toautoclaved soilrite (a mixture of horticulture-grade perlite with Irish peat moss and exfoli-ated vermiculite supplied by Kel Perlite, Bangalore, India) in bottles moistened with onefourth strength of MS basal salts. The bottles containing IBA-treated shoots on sterile soilritewere kept in the greenhouse near the pad section (high humidity and low temperaturecondition) at 80–90 % RH and 28±2 °C. After induction of roots, the caps of bottles weregradually opened over a period of 2 weeks and finally removed. Ex vitro-rooted plantletswere gradually shifted away from the pad section of the greenhouse towards fan section(temperature 32±2 °C and 60–70 % RH). After 25–30 days, the hardened plantlets weretransferred to poly bags containing clay, sand, and organic manure (1:1:1) and kept near thefan section. The hardened plantlets were shifted to the nursery.

Assessment of Genetic Fidelity Using DNA-Based Molecular Markers

The genetic fidelity among the mother plant and 14 randomly selected in vitro-regeneratedplants established in soil was assessed by polymerase chain reaction (PCR)-based RAPDand ISSR analysis. Genomic DNA was extracted from the fresh leaves of the mother plantand in vitro-regenerated plants according to CTAB method [24]. The concentration of DNAwas determined by spectrophotometer (UV-Visible Elico spectrophotometer) and quality ofgenomic DNAwas checked following electrophoresis on 0.8 % agarose gel. Genetic fidelityof tissue culture-raised plants was assessed using 15 RAPD and 7 ISSR primers. DNAamplification reactions for RAPD and ISSR markers was performed in a volume of 15 μlreaction mixture containing 2.0 μl template DNA (50–60 ng), 1.5 μl of 10× PCR buffer

Appl Biochem Biotechnol (2013) 170:1163–1173 1165


(Bangalore Genei, India), 1.5 μl of 2.5 mM MgCl2 (Bangalore Genei, India), 0.3 μl of10 mM dNTPs (Bangalore Genei, India), 0.3 μl Taq polymerase (five units, BangaloreGenei, India), and 1.0 μl (for RAPD) or 1.5 μl (for ISSR) of 10 μM primer (Integrated DNATechnologies Inc., India). DNA amplification was carried out in a thermal cycler (Eppendorf5331, Germany). The PCR program for RAPD consisted of an initial denaturation for 4 minat 94 °C, then 40 cycles of 30 s denaturation at 94 °C, 1.20 min annealing at annealingtemperature (Ta) (in degree Celsius), and 1.30 min extension at 72 °C with a final extensionat 72 °C for 10 min. The PCR for ISSR was performed as follows: initial denaturation at94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at Ta (indegree Celsius) for 60 s, and extension at 72 °C for 60 s with a final extension at 72 °C for10 min. The samples were stored at 4 °C until further processing. The Ta was kept 2 °Cbelow the Tm of that particular RAPD or ISSR primer sequence. Amplification with eachprimer was repeated twice to confirm reproducibility of the results. The amplificationproducts for all samples were resolved on 1.4 % agarose (A9539, Sigma, St Louis, MO,USA) gel using 1× TBE buffer and stained with ethidium bromide. Gels were visualizedusing a gel documentation system (Syngene Gel Doc, Syngene, Synoptics Ltd., UK). Thesize of the amplicons was estimated by comparing with 100 bp DNA ladder (Genei,Bangalore, India).

Experimental Design and Statistical Analysis

All the experiments were conducted with minimum of 20 replicates per treatment. Oneexplant represented one replicate. Each experiment was repeated three times. The results areexpressed as mean±SD of three experiments. Observations were recorded after 3–4 weeksof intervals. The data were analyzed statistically using one-way analysis of variance, and thesignificant differences between means were assessed by Duncan’s multiple range test at P<0.05. For molecular studies, only consistently reproducible and well-resolved bands, rangingfrom 200 to 1,200 (for RAPD and ISSR markers), were manually scored and the scoring ofbands was recorded in the form of their presence (“1”) or absence (“0”) in the gel.

Results and Discussion

Explant Selection, Shoot Induction, and Multiplication

In the present study, nodal segments with one to two nodes were used as explants for cultureestablishment. In comparison to nodal segments with one to two nodes, soft/apical shootsdid not give desirable results in terms of percent response and average shoot number. Similarto our observation, the beneficial response of nodal segments was also reported in a numberof plants like Salvadora persica [25], Salvadora oleoides [26], Terminalia bellirica [27], andTerminalia catappa [28]. Irrespective of cytokinin concentrations, nodal shoot segmentsshowed shoot bud induction on medium containing BAP or Kin. Out of all concentrations ofBAP/Kin tested, 2.0 mg l−1 BAP was optimal for shoot bud induction. On this medium,70.2 % explants produced 3.20±0.42 shoots per explant within 2–3 weeks (Table 1). Theexplants produced few shoots on higher or lower concentrations of BAP or Kin. At higherconcentrations (3.0–5.0 mg l−1) of BAP or Kin, a mass of callus was formed at base ofexplants resulting in reduced average shoot number and shoot length. Kin was found to beless effective cytokinin as compared to BAP. The effectiveness of BAP on multiple shootinduction has also been demonstrated in a number of cases [16, 29–31].



In vitro-regenerated shoots from explants were multiplied by transferring them on freshMS medium containing 0.5 mg l−1 of BAP; on this medium, the highest number (28.25±0.40) of shoots were obtained (Table 2). In order to further enhance the rate of shootmultiplication and elongation, shoot clumps were cultured on MS + 0.5 mg l−1 BAP withIAA or NAA. On MS medium containing 0.5 mg l−1 BAP + 0.1 mg l−1 IAA, maximum of47.50±0.38 shoots (each 5.20±0.10 cm long) per shoot clump was regenerated (Fig. 1a,Table 2). Enhancement of shoot multiplication by addition of low level auxins along withcytokinin has also been reported in many plant species like Rauvolfia tetraphylla [16],Momordica dioica [30], Ceropegia bulbosa [31], and Pseudarthria viscida [32].

Table 1 Effect of cytokinins (BAP or Kin) on shoot induction from nodal explants of A. hispidissima

BAP (mg l−1) Kin (mg l−1) Percentage of bud breaking Shoot number(mean±SD)

Shoot length (cm)(mean±SD)

0.0 0.0 0.0i 0.0e 0.0h

1.0 – 45.4f 1.20±0.41d 1.14±0.21e

2.0 – 70.2a 3.20±0.42a 2.21±0.44a

3.0 – 64.5b 2.52±0.12b 2.10±0.21b

4.0 – 60.3c 2.10±0.32bc 2.04±0.23b

5.0 – 55.5d 1.40±0.32d 1.42±0.27c

– 1.0 25.5j 1.20±0.41d 1.06±0.32fg

– 2.0 50.0e 1.45±0.23d 1.24±0.11d

– 3.0 40.5g 1.20±0.24d 1.12±0.28ef

– 4.0 40.0g 1.11±0.37d 1.08±0.30ef

– 5.0 35.3h 1.30±0.47d 1.01±0.32g

Medium: MS + additives. Means in each column followed by same letters are not significantly differentaccording to DMRT at P<0.05

Table 2 Effect of cytokinin (BAP) alone or combination of optimized concentration of BAP with auxins(IAA or NAA) for shoots multiplication and elongation of A. hispidissima

BAP (mg l−1) IAA (mg l−1) NAA (mg l−1) Shoot number (mean±SD) Shoot length (cm) (mean±SD)

0.0 0.0 0.0 0.0h 0.0g

0.25 – – 21.10±0.06fg 2.11±0.34f

0.5 – – 28.25±0.40d 4.19±0.40c

1.0 – – 24.54±0.22ef 3.22±0.44d

1.5 – – 21.40±0.30ij 2.10±0.20f

0.5 0.05 – 32.32±0.20bc 4.66±0.28b

0.5 0.1 – 47.50±0.38a 5.20±0.10a

0.5 0.2 – 34.10±0.41b 4.20±0.20c

0.5 0.3 – 29.12±0.20cd 3.46±0.38d

0.5 – 0.05 21.64±0.36fg 3.11±0.35de

0.5 – 0.1 27.20±0.41de 2.70±0.20e

0.5 – 0.2 22.52±0.10fg 2.56±0.28e

0.5 – 0.3 19.14±0.42g 2.54±0.34e

Medium: MS + additives. Means in each column followed by same letters are not significantly differentaccording to DMRT at P<0.05



Ex Vitro Rooting and Acclimatization of In Vitro-Produced Plantlets

The in vitro-regenerated shoots treated with 300 mg l−1 of IBA (for 5 min) exhibited about90 % rooting with maximum of 3.42±0.20 roots per shoot and 3.25±0.27 cm root lengthwithin 3–4 weeks (Table 3, Fig. 1b). IBA is more prominent than NAA in promoting rootingof a wide variety of plants, and it is used commercially for rooting of many plant species [16,28, 33]. Ex vitro rooting is not only helpful in the reducing resources, time, and labor costbut also simplifies the protocol by eliminating the rooting step under sterile conditions, anadditional step of micropropagation [34–36]. Furthermore, it has been also reported that exvitro-rooted plants are better suited to tolerate environmental stresses [26, 27, 37]. Theplantlets developed from ex vitro rooting were successfully acclimatized and established insoil with a survival rate of 85–90 % (Fig. 1c, d).

Assessment of Genetic Fidelity of Micropropagated Plants

The plants raised through in vitro propagation were evaluated for their genetic fidelity byDNA-based molecular markers. In the present study, the use of two PCR-based markers, i.e.,RAPD and ISSR, were selected because of their simplicity, cost-effectiveness, quickness in

Fig. 1 Micropropagation of A. hispidissima. a Multiplication of shoots on MS + 0.5 mg l−1 BAP + 0.1 mg l−1

IAA. b Ex vitro-rooted shoot after treatment with 300 mg l−1 IBA. c Hardened plants developed from ex vitrorooting in screw bottles. d Hardened plant transferred to the pot



Table 4 DNA-based molecular marker for assessment of genetic fidelity of micropropagated plants of A.hispidissima

Primer code Primer sequence No. of scorable bands Range of amplification (bp)

RAPD

OPC-02 GTGAGGCGTC 4 200–1,000

OPA-13 CAGCACCCAC 5 300–1,100

OPH-05 AGTCGTCCCC 4 300–1,000

OPG-07 GAACCTGCGG 6 400–1,100

OPAB-18 CTGGCGTGTC 5 300–1,200

OPB-07 GGTGACGCAG 4 300–1,100

OPQ-07 CCCCGATGGT 8 300–1,000

OPA-15 TTCCGAACCC 4 300–1,000

OPA-14 TCTGTGCTGG 5 300–1,200

OPC-01 TTCGAGCCAG 6 200–1,200

OPN-13 AGCGTCACTC 6 400–1,100

OPAB-06 GTGGCTTGGA 4 200–1,000

OPA-04 AATCGGGCTG 5 300–1,000

OPB-03 CATCCCCCTG 5 300–1,100

OPA-06 GGTCCCTGAC 6 300–1,200

ISSR

UBC-822 TCTCTCTCTCTCTCTCA 5 200–800

UBC-823 TCTCTCTCTCTCTCTCC 6 400–1,200

UBC-824 TCTCTCTCTCTCTCTCG 4 200–1,000

UBC-835 AGAGAGAGAGAGAGAGYC 5 200–1,000

UBC-843 CTCTCTCTCTCTCTCTRA 4 300–1,200

UBC-844 CTCTCTCTCTCTCTCTRC 4 300–1,100

UBC-845 CTCTCTCTCTCTCTCTRG 6 300–1,000

Table 3 Effect of auxins (IBA or NAA) on ex vitro rooting of micropropagated shoots of A. hispidissima

IBA (mg l−1) NAA (mg l−1) Percentage of rooting Root number(mean±SD)

Root length (cm)(mean±SD)

0.0 0.0 0.0h 0.0h 0.0k

100 – 50.2e 1.31±0.31e 2.05±0.44cd

200 – 70.0c 2.25±0.32c 2.55±0.16c

300 – 90.0a 3.42±0.20a 3.25±0.27a

400 – 81.3b 2.75±0.32b 2.87±0.21b

500 – 60.5d 1.78±0.11d 1.65±0.22f

– 100 40.2f 1.11±0.33fg 1.10±0.20hij

– 200 60.6d 1.80±0.41d 2.10±0.30de

– 300 48.5e 1.25±0.34ef 1.62±0.22fg

– 400 36.4fg 1.02±0.21g 1.08±0.32hi

– 500 31.8g 1.10±0.43fg 1.01±0.46h

Treatment duration, 5 min. Means in each column followed by same letters are not significantly differentaccording to DMRT at P<0.05



M P 1 2 3 4 5 6 7 8 9 10 11 12 13 14

a

M P 1 2 3 4 5 6 7 8 9 10 11 12 13 14

b

Fig. 2 DNA amplification obtained with RAPD primers a OPG-07; b OPQ-07. M, DNA marker(ladder 100 bp); P, DNA from mother plant; 1–14, DNA from micropropagated plants

M P 1 2 3 4 5 6 7 8 9 10 11 12 13 14

M P 1 2 3 4 5 6 7 8 9 10 11 12 13 14

a

b

Fig. 3 DNA amplification obtained with ISSR primers a UBC-823; b UBC-845. M, DNA marker (ladder100 bp); P, DNA from mother plant; 1–14, DNA from micropropagated plants



operation, and requirement of small quantity of DNA [11, 38]. Out of the 25 RAPDprimers screened, only 15 resulted in four to eight scorable bands per primer. These15 RAPD primers generated 77 amplicons in total, ranging from 200 to 1,200 bp insize. The number of bands in the selected primers varied from four (OPC-02, OPH-05, OPB-07, OPA-15, and OPAB-06) to eight (OPQ-07), with an average of 5.13bands per RAPD primer (Table 4). Out of the 15 ISSR markers screened, only 7primers resulted in four to six scorable bands, which were selected for further study.These 7 ISSR primers generated 34 amplicons, ranging from 200 to 1,200 bp in size.The number of bands for each primer varied from four (UBC-824, UBC-843 andUBC-844) to six (UBC-823 and UBC-845), with an average of 4.86 bands per ISSRprimer (Table 4). A total of 15 RAPD and 7 ISSR primers generated 111 distinctamplicons. RAPD (Fig. 2a, b) and ISSR (Fig. 3a, b) banding patterns of in vitro-regenerated plants were monomorphic and similar to those of the mother plantconfirming their genetic fidelity. Explant types, mode of regeneration, and tissueculture conditions cause various genetic changes in regenerated plants; however, directplant regeneration through organized tissues like meristems generally avoid geneticchanges and have lower chance of genetic variability [9–12]. Our results confirm thereports of Rathore et al. [10] in Aloe vera, who suggested that the use of shoot budexplants generally maintains genetic stability of the plantlets obtained through tissueculture with a least risk of producing somaclonal variants. Assessment of geneticstability of in vitro-raised plantlets simultaneously by RAPD and/or ISSR markers hasalso been reported in many other plants like A. vera [10], Simmondsia chinensis [14],Psidium guajava [15], R. tetraphylla [16], Dendrocalamus asper [17], Capparisspinosa [39], and Celastrus paniculatus [40].

Conclusion

The present study describes an improved and efficient plant regeneration method for A.hispidissima through axillary shoot bud proliferation. Monomorphic banding patternobtained among mother plant and tissue culture-raised plants of A. hispidissima with twoDNA-based markers confirmed the genetic stability of the plants produced and also approvethe commercial scale utilization of the developed protocol.

Acknowledgments The authors (MP, MKR) wish to acknowledge the support of the University GrantsCommission, NewDelhi, for the award of Post Doctoral Fellowship and Dr. DSKothari Post Doctoral Fellowship,respectively. We thank Department of Biotechnology, Government of India, New Delhi, for providing funds forthe establishment of laboratory and greenhouse infrastructure used for the present research.

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