ORIGINAL ARTICLE

Rapid and stable in vitro regeneration of plants through callusmorphogenesis in two varieties of Mucuna pruriens L. – an antiParkinson’s drug yielding plant

Kotisree Lahiri & Madhumita J. Mukhopadhyay &

Yves Desjardins & Sandip Mukhopadhyay

Received: 31 January 2012 /Published online: 12 May 2012# Archana Sharma Foundation of Calcutta 2012

Abstract Mucuna pruriens L. is an important medicinalplant reported to contain L-3,4-dihydroxy phenylalanine(L-DOPA). The present report describes a simple protocolfor plant regeneration through callus morphogenesis in fourstrains belonging to two different varieties of Mucuna pru-riens. Friable, soft and nodular callus was induced fromnodal and internodal segments of in vitro-grown seedlingson modified Murashige and Skoog’s (MS) medium supple-mented with 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine(BAP). For regeneration of shoots, the callus tissues weretransferred to medium supplemented with various levels ofcytokinin. Highest shoot regeneration from the callus tissuewas achieved in modified MS medium fortified with BAP at1.33 μM level. The regenerated shoots were rooted in vitroin half-strength of liquid MS medium supplemented withvarious levels of NAA. The regenerates were acclimatizedfor 2–3 weeks and showed about 80 % survival rate aftertransferring to the field. Cytological analysis revealed chro-mosome number stability of all the regenerates (2n022)with complete absence of aneuploidy. Random Amplified

Polymorphic DNA (RAPD) and Inter Simple SequenceRepeat (ISSR) analyses of total genomic DNA supportedstability at the molecular level among all the clones of thesestrains when compared with their mother plants.

Keywords Callus morphogenesis . Chromosome . Geneticfidelity . ISSR .Mucuna pruriens . Plant regeneration .

RAPD

Introduction

Mucuna, a genus of annual and perennial twining herbs orshrubs of the Fabaceae family, contains about 15 species inIndia [31]. Mucuna pruriens L., commonly known as velvetbean, is an important medicinal plant growing in the bushes,hedges and dry deciduous forests throughout India [1, 26,29]. Two varieties of M. pruriens (vars. pruriens and utilis)are found in India.M. pruriens var. pruriens is found in wildwhereas M. pruriens var. utilis is a cultivated variety. Thesetwo varieties differ in seed and pod characteristics. Since allparts ofM. pruriens are reported to contain L-3,4-dihydroxyphenylalanine (L-DOPA) [3], a neurotransmitter precursor,used in the treatment of Parkinson’s disease, there is an ever-increasing demand of Mucuna in the international drugmarket. Large-scale extraction of L-DOPA from the wildpopulations of this plant has led to its limited availability innatural condition.

The plant usually propagates through seeds and, thegermination rate and viability of seeds are very poor. There-fore, in vitro plant culture techniques including organ cul-ture and callus culture [14] may be an effective alternativefor propagation and conservation of plants of such an eco-nomic importance in which conventional methods show lim-itations. The development and improvement of procedures

K. Lahiri : S. Mukhopadhyay (*)Centre of Advanced Study, Department of Botany,University of Calcutta,35, Ballygunge Circular Road,Kolkata 700019, Indiae-mail: sanmukh2000@hotmail.com

M. J. MukhopadhyayDepartment of Biotechnology, Institute of Genetic Engineering,30, Thakurhat Road, BADU,Kolkata 700128, India

Y. DesjardinsCentre de recherche en horticulture, Pavillon de l’Envirotron,Université Laval,Québec, QC G1V 0A6, Canada

Nucleus (April 2012) 55(1):37–43DOI 10.1007/s13237-012-0051-7

for efficient regeneration of plants from cultured cells, tissuesand organs are necessary for application of in vitro culturetechniques to plant gene manipulation for crop germplasmenhancement [32]. Depending on the composition of thecallus culture medium, plant regeneration in culture can occurby either of the two possible routes, i.e. organogenesis andsomatic embryogenesis. There are reports on micropropaga-tion of M. pruriens [4, 8–10, 12, 13, 27, 30] but callusmorphogenesis for in vitro propagation has not yet beenreported for this species. The present study describes a simpleand efficient method for plant regeneration in culture viacallus morphogenesis/organogenesis in both the varieties ofM. pruriens L. (vars. pruriens and utilis), available in India. Inthe present report, the genetic fidelity of the regenerates hasbeen tested using both cytological and molecular analyses.

Materials and methods

Plant materials

Seeds of three strains of Mucuna pruriens var. utilis [StrainNos.: IC 471876 (Strain I), IC 241679 (Strain II), IC 392338(Strain III)] were obtained from NBPGR, New Delhi. Plantsof one strain of Mucuna pruriens var. pruriens (Strain IV)were collected in wild from West Midnapur district of WestBengal, India.

Culture conditions

Seeds of the four strains of M. pruriens were surface steril-ized by treating with 0.1 % (w/v) aqueous mercuric chloride(HgCl2) solution for 15 min followed by thorough washingin sterile distilled water. The seeds were then cultured onMS [22] basal medium containing 3 % (w/v) sucrose, and0.25 % (w/v) Gelrite®, adjusted to pH of 5.7 and withoutany exogenous growth regulator. Modified MS basal mediawith 3 % (w/v) sucrose, 0.1 g/L each of glutamine andascorbic acid along with various concentrations and combi-nations of different growth regulators were used for callusinduction. The pH of the callus induction medium wasadjusted to 5.8, while that of the callus regeneration mediumwas adjusted to 5.7 prior to gelling with 0.25 % (w/v)Gelrite®. The cultures were incubated in a culture room at24°C±1°C and with 55 % maximum relative humidity,under a photoperiod of 16 h supplied by cool white fluores-cent tubes providing a light intensity of 48 μmol m−2 s−1.

Callus induction and regeneration

The nodal and internodal segments of the 7-day old in vitrogrown seedlings were cultured on modified MS mediumsupplemented with various concentrations and combinations

of auxins (2,4-D and NAA) and cytokinins [BAP, 6-(γ,γdimethylallyl amino)-purine (2iP) and kinetin (Kn)].

For regeneration from callus tissue, the nodular, soft andfriable callus masses obtained in the medium containing2,4-D (4.5 μM), NAA (5.4 μM) and BAP (0.88 μM) weretransferred to modified MS medium fortified with 0.88–4.44 μM BAP or 0.93–4.65 μMKn. The regenerating callusmasses were sub-cultured at 4 weeks interval in the sameregeneration media.

In vitro rooting

The regenerated plants were cultured on full strength MSbasal medium, half-strength MS basal medium (both liquidand semi-solid) and also on modified half-strength MS basalmedium (both liquid and semi-solid) supplemented withvarious levels of either NAA (0.54–10.7 μM) or indole-3-butyric acid (IBA) (0.49–9.85 μM).

Acclimatization of the regenerates

The completely developed plants were potted in sterileSoilrite® mixture (mixture of horticulture grade expandedperlite, Irish Peat moss and exfoliated vermiculite in equalratio) at 24±1°C, with spraying of MS salts solution everyalternate day to acclimatize for 2–3 weeks. These were thentransferred to soil.

Clonal fidelity testing

Chromosome analysis from root tip cells of the regenerateswas carried out following acetic-orcein squash schedule [19]to determine ploidy status. RAPD and ISSR studies weredone to assess the fidelity of the regenerated clones. ForISSR and RAPD analysis, leaf samples were collected frommother plant and five randomly selected tissue culturedclones of each strain. DNA from young leaves was extractedusingmodified Doyle and Doyle [7] method. To the extractionbuffer containing cetyltrimethylammonium bromide (CTAB),0.5 % charcoal along with 0.2 % β-mercaptoethanol wasadded to avoid polyphenol oxidation. After propanol precipi-tation, DNA was re-suspended in 1X Tris-EDTA buffer(pH 8.0) and quantified spectrophotometrically by taking theabsorbance at 260 nm. ISSR and RAPD assays were carriedout in 25 μl reaction mixture containing 1X Taq DNA poly-merase buffer [75 mM Tris–HCl (pH 8.8), 20 mM(NH4)2SO4], 0.2 mM dNTP mix (Fermentas), 3 mM MgCl2,0.5 μM primer (Bangalore Genei), 1 U Taq DNA polymerase(Fermentas), and 50 ng template DNA. PCR reactions werecarried out in a thermocycler (Eppendorf Master Cycler Gra-dient). Reactions without DNAwere used as negative control.For both ISSR and RAPD studies, the thermocycler wasprogrammed for an initial denaturation step of 4 min at

38 Nucleus (April 2012) 55(1):37–43

94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 36°C(for RAPD) or 1 min at 55°C (for ISSR) and extension at 72°Cfor 2 min. The last cycle was followed by a final extension at72°C for 10 min and a hold temperature of 4°C at the end.Amplified products were resolved in 1.4 % agarose gel(1x TBE) followed by ethidium bromide staining. Gene Rulerfrom Fermentas was used as marker.

Statistical analysis

Each treatment was repeated 3 times with 5 replicates ofeach. Data were recorded at 30 days interval up to 150 days.The square root transformations of the data were submittedto an analysis of variance (ANOVA) using the softwareSPSS 16, to detect significant differences between means

Table 1 In vitro responses of different growth regulators on callusinduction of Mucuna pruriens L.

MS+GR (μM) Response Type of callus formed

NAA (10.75)+BAP (0.88) +++ Soft, green, nodular, profusegrowth

NAA (5.4)+Kn (0.93) ++ Soft, green, slow growth

NAA (5.4)+2iP (1.0) ++++ Very hard, compact, yellowishgreen, massive growth

2,4-D (9.0)+BAP (0.88) ++++ Greenish-white, soft, nodular,profuse growth

2,4-D (4.5)+NAA (5.4)+ BAP (0.88)

+++++ Creamish white, soft, nodular,embryogenic, huge growth

NAA (10.75)+BAP(4.44)

+++ Slightly hard, green, profusegrowth

‘+’ denotes the degree of response

Fig. 1 Callus morphogenesis and plant regeneration in Mucuna pru-riens L. (bar05 mm): (a) The induced callus tissue in MS+5.4 μMNAA+4.5 μM 2,4-D+0.88 μM BAP, (b) Regeneration from callustissue in modified MS medium+1.33 μM BAP, (c) Rooting of the

regenerates, (d) Acclimatized regenerated plants, (e) Somatic meta-phase plate from root tips of the regenerate showing 2n022 chromo-somes (bar010 μm)

Nucleus (April 2012) 55(1):37–43 39

[12]. To ensure normality and equal variance among groups,the square root transformation of the data was used. Squareroot transformation for count data is one of the applicationsof the general data transformation theory [25].

Results and discussion

Callus formation was observed in both nodal and internodalsegments at the cut ends of explants. However, callus for-mation was also noticed lately just below the nodes after aperiod of swelling. The responses of the strains to differentmedia varied with the growth regulator combinations. How-ever, among the different strains, the nature of response to aparticular growth regulator combination was more or lesssimilar, though the proliferation rate differed. Callus wasformed after 10 days in culture in the medium containingtwo auxins with a low level of cytokinin (5.4 μM NAA+4.5 μM 2,4-D+0.88 μM BAP). Callus formed in this me-dium, in all the four strains, was creamish-white in colour,friable and nodular in nature, and proliferated very rapidly(Table 1; Fig. 1a). In all the four strains studied, 2,4-D(9.0 μM) in combination with BAP (0.88 μM) was also

effective for induction of soft and nodular callus tissue.However, the callus proliferation rate was less on this me-dium than the medium containing two auxins (NAA and2,4-D) with a low level of BAP. In the media containingNAA (10.75 μM) and BAP (0.88 μM), the callus formedwas soft, nodular and green in colour. Kinetin and 2-iP(0.93 μM and 1.0 μM respectively) used individually inplace of BAP in association with the same concentrationof NAA did not induce formation of soft, friable and nodularcallus (Table 1). In the media containing NAA and 2iP, amassive callus was formed after about 10 days in culture.However, the induced callus was greenish-yellow, highlyproliferating but extremely hard and compact. Kinetin incombination with NAA, on the other hand, was not as effec-tive as BAP in induction of callus tissue and the proliferationrate was also quite low. NAA (10.75 μM) in combinationwith a higher concentration of BAP (4.44 μM) produced aslightly hard and green callus, in all the four strains (Table 1).Soft, friable, nodular and highly proliferating callus tissuewas induced in the media containing two auxins and a cyto-kinin in all these strains and this may be due to the synergisticeffect of the two auxins [24] in callus induction.

The white, soft, friable and nodular proliferating callustissues (produced in medium containing NAA, 2,4-D andBAP) were transferred to callus regeneration media. It wasfound that the elimination of auxin from these mediaresulted in the distribution of certain regions showing green,protruding meristemoids on the callus surface after 15–20 days in culture. However, the nodal callus was moreresponsive to regeneration than the internodal callus. Inthese regions, green primordia-like structures developedinto small shoot-buds after transferring to fresh medium(Fig. 1b). Among the two cytokinins, BAP was found tobe more effective for callus organogenesis than kinetin. Theconcentration, 1.33 μM of BAP induced the highest numberof shoot buds in all these four strains. The only strain ofvariety pruriens produced highest number of shoot buds(16.6) as compared to all other three strains of variety utilis.

Table 2 Three-way ANOVA with multiple observations in each cate-gory on shoot bud regeneration from callus tissue in four differentstrains of Mucuna pruriens L.

Source DF F value P value

Time 4 154.87 0.0001

Treatment 7 148.79 0.0001

Strain 3 2.89 0.0347

Time*Treatment 28 5.65 0.0001

Time*Strain 12 0.17 0.9993

Treatment*Strain 21 4.21 0.0001

Time*Treatment*Strain 84 0.15 1.0000

Error 640

Table 3 Responses of differentconcentrations of growth regu-lators on shoot bud regenerationfrom callus tissue of Mucunapruriens L.

Modified MS medium+GR [μM] Mean number of shoot buds in four strains of M. pruriens L.

30 days 60 days 90 days 120 days 150 days

BAP [0.88] (Treatment 1) 0.65 3.1 5.5 8.05 9.2

BAP [1.33] (Treatment 2) 2.05 4 9.75 11.75 13

BAP [2.22] (Treatment 3) 0.35 1.35 2.35 2.8 3.3

BAP [4.44] (Treatment 4) 0.65 1.05 1.5 2 2.4

Kinetin [0.93] (Treatment 5) 0.6 1.1 1.5 1.8 2.2

Kinetin [1.39] (Treatment 6) 0.55 1.15 2.1 3.3 3.65

Kinetin [2.32] (Treatment 7) 0.55 0.95 1.75 2.2 2.55

Kinetin [4.65] (Treatment 8) 0.25 0.5 0.95 1.2 1.5

LSD values 0.3965 0.5532 0.8756 1.0363 1.1557

40 Nucleus (April 2012) 55(1):37–43

Among the variety utilis, strain II produced the highestnumber of shoot buds (15.2), whereas the least was recordedin strain III (9.4) after 150 days in culture. The rate ofincrement in the number of shoot buds produced was high-est from 60 days to 90 days in culture. A gradual incrementin number of shoot buds was observed after 90 days inculture. The ANOVA table shows that the responses of the

plants to the different treatments are highly significant(Table 2). From the ANOVA table, it is also observed thatthe interactions of time*treatment and strain*treatment aresignificant (Table 2). This may be due to the fact that theresponses of all the four strains were not the same. However, theinteraction between time*treatment*strain is not significantindicating that all treatments behaved the same notwithstanding

Table 4 RAPD and ISSR analyses of the regenerated clones of Mucuna pruriens L.

Primer Type Primer Name Sequence (5′–3′) Results Size range (bp) Range of scorable bands(among 4 strains)

ISSR I 01 (CA)8AT Positive, reproducible, monomorphic 290–1200 4–5

ISSR I 03 (CA)7CC Positive, reproducible, monomorphic 210–890 8–10

ISSR I 09 (GA)8T Positive, not reproducible _ _

ISSR I 15 (TG)7C Negative _ _

ISSR I 20 (TC)8AG Positive, reproducible, monomorphic 225–1050 8–9

RAPD B 08 TCACCACGGT Positive, reproducible, monomorphic 325–1250 5–7

RAPD B 31 ATTCAGTCAC Positive, not reproducible _ _

RAPD OPA 07 GAAACGGGTG Positive, reproducible, monomorphic 190–1930 5–10

RAPD OPA 16 AGCCAGCGAA Positive, not reproducible _ _

RAPD OPA 20 GTTGCGATCC Positive, not reproducible _ _

Fig. 2 Agarose gel electrophoresis of amplified fragments of in vitro-regenerated clones (1–5) with their respective mother plants (P1–P4) showingmonomorphic bands generated by the ISSR primer I 20 in (a) Strain I, (b) Strain II, (c) Strain III, (d) Strain IV. Lane M-Molecular weight marker

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the time and the strain (Table 2). The LSD analysis (Table 3)shows that the response of the strains in the media containing1.33 μM BAP is significantly different from the responses tothe other media studied during the entire period of the study.

Several factors including physical, chemical, physiolog-ical and cytological have been found to control morphogen-esis. The use of appropriate plant growth regulator at adefinite concentration and optimum culture conditions areimportant for successful plant regeneration in culture. Therole of phytohormone is well established in both organo-genesis and embryogenesis processes [28] and in somespecies the differentiation process is governed by quantita-tive interactions between the different hormones [6]. In thepresent study, BAP has been found to be more effective thankinetin in callus morphogenesis. Similar reports on efficacy ofBAP over kinetin are also available for other plant species [2,15, 17, 18]. The shoots, after attaining a length of 2.0–3.0 cm,were transferred to a medium without any growth regulator.The induction of roots from the regenerated shoots in presenceof low concentrations of MS medium (half-strength) supple-mented with only NAA was suitable in this study. MS basalmedium without any growth regulator showed a delayed andweak response. Though root formation from regeneratedshoots was induced at low IBA concentration (0.98 μM),higher concentrations of IBA (4.9 μM and 9.8 μM) werefound to be less effective in this process. On the other hand,NAA at lower concentrations (0.54 μMand 1.07μM)was notvery effective for in vitro root induction. Large numbers ofstout and healthy roots were formed at 5.4 μM NAA, after2 weeks in culture, in all the strains studied (Fig. 1c). How-ever, further increase in NAA concentration did not favour invitro rooting. The efficiency of NAA in root induction fromshoots in vitro has been reported for several plant species [5,23] including Mucuna pruriens [12, 27].

The acclimatized plants were transferred to field andshowed~80 % survival rate. The high survival rate of thecallus regenerates in the field also indicated the superiorityof anatomical and morphological nature of the regeneratedroots and the suitability of both physical and chemicalenvironments in culture (Fig. 1d). It might, therefore, besuggested that organ differentiation in culture might havebeen the result of optimization of both type and concentra-tion of exogenous growth regulators [20]. Moreover, thegenome specificity between these strains of M. pruriens,which is under genetic control, was reflected in their in vitroresponses during callus regeneration.

Chromosome analysis was carried out on five randomlyselected in vitro regenerated plants of each of the fourstrains of Mucuna pruriens. Chromosome number stabilitywas observed in all the strains. All the plants tested showed2n022 chromosomes (Fig. 1e), as also reported in the in vivoplants as well as in the regenerates developed through othermodes of regeneration [11–13]. The plant morphological

similarity and chromosome analysis indicated stability of theregenerates. ISSR and RAPD analysis were done in order toassess the genetic integrity of tissue culture clones. RAPDpattern of five randomly selected micropropagated plants ofeach strain were compared with the mother plant. Of the fiveRAPD and five ISSR primers screened, two RAPD and threeISSR primers yielded clear, reproducible bands. The numberof amplified bands for each primer varied from 04 in I 01(strain III) to 10 in OPA 07 (strains II and III) and I 03 (strainII). Each primer produced amplification products in the sizerange 0.19 kb to 1.93 kb (OPA 07) (Table 4). It was observedthat the RAPD and ISSR profiles of different micro-propagated clones were typical to that of the donor motherplant (Figs. 2a–d). It may be suggested that the culture con-ditions did not affect the genetic make-up during in vitroregeneration. This may indicate the presence of high numberof potential diploid cells during regeneration. Chromosomenumber stability in culture is considered to be most essentialfor clonal propagation [16]. In the present study, however, noaneuploidy was observed in the regenerates of all the fourstrains. This may be due to the fact that the cells duringregeneration were not exposed for long period to auxins. Also,the type and concentration of exogenous auxin, as well as theduration of culture might have been responsible for suchstability [21]. In the present study, auxin has been utilized onlyfor callus induction and cultured only for a few weeks. Theregeneration medium contained only a single cytokinin with-out any auxin which might have been responsible for main-taining genomic stability of the regenerates.

The present study describes an efficient and simple proto-col of callus morphogenesis for clonal propagation of twovarieties of M. pruriens with genomic stability, both at chro-mosome and molecular levels. It is less work intense thanmicropropagation and provides high yield proliferation. Onecould foresee the adaptation of this protocol in a biofermentorsystem for mass propagation of true to type plants.

Acknowledgements The financial assistance from University GrantsCommission, New Delhi, [MRP. No. F. 32-415/2006 (SR)] is grateful-ly acknowledged. The authors gratefully acknowledge the help offeredby Mr. Bikash Kundu for data analysis. The authors also wish toacknowledge NBPGR, New Delhi for supplying seeds of Mucunapruriens var. utilis.

References

1. Agharkar SP. Medicinal plants of Bombay presidency. ScientificPub 1, Jodhpur, India; 1991. pp. 1–2.

2. Benmoussa M, Mukhopadhyay S, Desjardins Y. Optimization ofcallus culture and shoot multiplication of Asparagus densiflorus.Plant Cell Tiss Org Cult. 1996;47:91–4.

3. Caius JF. The medicinal and poisonous legumes of India. ScientificPub 1, Jodhpur, India; 1989. pp. 70–71.

42 Nucleus (April 2012) 55(1):37–43

4. Chattopadhyay S, Datta SK, Mahato SB. Rapid micropropa-gation for Mucuna pruriens f. pruriens L. Plant Cell Rep.1995;15:271–3.

5. Chithra M, Martin KP, Sunandakumari C, Madhusoodanan PV.Silver nitrate induced rooting and flowering in vitro on rare rhoeo-phytic woody medicinal plant, Rotula aquatica Lour. Indian JBiotech. 2004;3:418–21.

6. Desjardins Y. Micropropagation of Asparagus officinalis L. In:Bajaj YPS, editor. High-tech and micropropagation III. Biotech-nology in agriculture and forestry, Vol 19. Berlin Heidelberg NewYork: Springer; 1992. p. 26–41.

7. Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue.Focus. 1990;12:13–5.

8. Faisal M, Siddique I, Anis M. An efficient plant regenera-tion system for Mucuna pruriens L. (D.C.) using cotyledon-ary node explants. In Vitro Cell Dev Biol Plant. 2006;42:59–64.

9. Faisal M, Siddique I, Anis M. In vitro rapid regeneration ofplantlets from nodal explants of Mucuna pruriens – a valuablemedicinal plant. Ann Appl Biol. 2006;148:1–6.

10. Harini SS, Sathyanarayana N. Somatic embryogenesis in Mucunapruriens. African J Biotech. 2009;8:6175–80.

11. Lahiri K, Mukhopadhyay MJ, Mukhopadhyay S. Karyotype anal-ysis and in situ 4 C nuclear DNA quantification in two varieties ofMucuna pruriens L. J Trop Med Plants. 2010;11:219–25.

12. Lahiri K, Mukhopadhyay MJ, Mukhopadhyay S. Enhancement ofL-DOPA production in micropropagated plants of two differentvarieties of Mucuna pruriens L., available in India. Plant Tiss CultBiotech. 2011;21:115–25.

13. Lahiri K, Mukhopadhyay MJ, Mukhopadhyay S. Simple and effi-cient plant regeneration following somatic embryogenesis inMucuna pruriens var. pruriens, a natural source of L-DOPA. JTrop Med Plants. 2012;13 (in press).

14. Lahiri K, Mukhopadhyay S, Mukhopadhyay MJ. A new reporton induction of embryogenic callus culture in Mucuna pruri-ens L., an important medicinal plant. J Bot Soc Beng.2006;60:1–4.

15. Lee SY, Kim YK, Uddin MR, Park NI, Park SU. An efficientprotocol for shoot organogenesis and plant regeneration of buck-wheat (Fagopyrum esculentum Moench.). Rom Biotech Let.2009;14:4524–9.

16. May RA, Sink KC. Genotype and auxin influence direct somaticembryogenesis from protoplast derived from embryogenic cellsuspensions of Asparagus officinalis L. Plant Sci. 1995;108:71–84.

17. Mukhopadhyay MJ, Lahiri K, Mukhopadhyay S. In vitro micro-tuberization and enhanced colchicine accumulation in two speciesof Gloriosa. Cytologia. 2008;73:357–63.

18. Mukhopadhyay MJ, Mukhopadhyay S, Sen S. In vitro propagationof Iphigenia indica, an alternative source of colchicine. Plant CellTiss Org Cult. 2002;69:101–4.

19. Mukhopadhyay S, Banerjee N. Chromosome study and karyotypeanalysis in Solanum sarrachoides Sendt. A new report. Cytologia.1989;54:179–82.

20. Mukhopadhyay S, Desjardins Y. A comparative study on plantregeneration from protoplasts of two genotypes of Asparagusofficinalis L. Plant Sci. 1994;100:97–104.

21. Mukhopadhyay S, Desjardins Y. Plant regeneration fromprotoplast-derived somatic embryos of Asparagus officinalis L. JPlant Physiol. 1994;144:94–9.

22. Murashige T, Skoog F. A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473–97.

23. Rauf S, Usman M, Fatima B, Khan TM, Khan IA. In vitro regen-eration and multiple shoot induction in upland cotton (Gossypiumhirsutum L.). Int J Agri Biol. 2004;6:704–7.

24. Rout JR, Sharma NP. Anther callus induction and green plant regen-eration at high frequencies from an interspecific rice hybrid Oryzasativa Linn. x O. rufipogon Griff. Euphytica. 1991;54:155–9.

25. Sakia RM. The Box-Cox transformation technique: a review. TheStatistician. 1992;41:169–78.

26. Sastry CST, Kavathekar YY. Plants for reclamation of wastelands.New Delhi: Publications and Information Directorate, CSIR; 1990.p. 317–8.

27. Sathyanarayana N, TN Bharath kumar, Vikas PB, Rajesha R. Invitro clonal propagation of Mucuna pruriens var. utilis and itsevaluation of genetic stability through RAPD markers. African JBiotech. 2008;7:973–80.

28. Sheridan WF. Plant regeneration and chromosome stability intissue culture. In: Ledoux L, editor. NATO Advanced Study Insti-tute on Genetic-Manipulation with Plant Material. New York:Plenum Press; 1975. p. 263–95.

29. Singh U, Wadhani AM, Johri BM. Dictionary of economic plantsin India. New Delhi: Indian Council of Agricultural Research;1996. p. 45–146.

30. Vibha JB, Choudhary K, Singh M, Rathore MS, Shekhawat NS.An efficient somatic embryogenesis system for velvet bean[Mucuna pruriens (L.) DC.]: a source of anti Parkinson’s drug.Plant Cell Tiss Org Cult. 2009;99:319–25.

31. Wealth of India: A dictionary of Indian Raw Materials and Indus-trial Products Vol VI, CSIR Publication, New Delhi; 1962. pp.439–444.

32. Zhang CL, Chen DF, Elliott MC, Slater A. Efficient procedures forcallus induction and adventitious shoot organogenesis in sugarbeet (Beta vulgaris L.) breeding lines. In Vitro Cell Dev Biol Plant.2004;40:475–81.

Nucleus (April 2012) 55(1):37–43 43