ijmap_4_1_7_solanum_viarum

Int. J. Med. Arom. Plants, ISSN 2249 4340RESEARCH ARTICLEVol. 4, No. 1, pp. 48-55, March 2014

*Corresponding author: (E-mail) challagundlav yahoo.co.in http://www.openaccessscience.com 2014 Copyright by the Authors, licensee Open Access Science Research Publisher. [email protected] is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0) License (http://creativecommons.org/licenses/by-nc-nd/3.0)

Efficient protocol for direct shoot organogenesis from in vitro raisednodal explants of Solanum viarum (Dunal) - An important anticancermedicinal plantM.D. MAHADEV, CHANDRA SEKHAR PANATHULA, C.V. NAIDU*

Department of Biotechnology, Dravidian University, Kuppam-517426, A. P., India*Corresponding author: Phone: +91-8772260386; Fax: +91-8570278209

Article History: Received 12th March 2014, Revised 25th March 2014, Accepted 27th March 2014.

Abstract: In the present study an attempt has been made to standardize a protocol for rapid direct plant regeneration fromin vitro derived nodal explants of S. viarum, which promoted shoot bud regeneration and resulted in maximum number ofmultiple shoot induction in the medium supplemented with different concentrations of plant growth regulators such ascytokinins like BAP, Kn alone or in combination with auxins like NAA/IBA/IAA. Among the combinations tested BAP(2.0 mg l-1) and IBA (0.5 mg/l-1) produced maximum number of shoots (22.30.41) per explant. The maximum shootlength (8.20.14 cm) was observed with Kn (1.0 mg l-1) and IBA (0.5 mg l-1) combination. All the in vitro raised shootswith a length of 3.0-5.0 cm were transferred to rooting medium supplemented with different concentrations of auxinssuch as NAA, IBA and IAA (0.2 1.0 mg l-1). The best rooting response was observed on IBA (0.6 mg l-1). The wellrooted plantlets were transferred to polycups containing soil and vermiculite in 1: 1 ratio for hardening. Finally the hard-ened plantlets were transferred to field conditions for maximum survivability.

Keywords: Multiple shoot induction; Nodal explants; Plant regeneration; Solanm viarum.

Abbreviations: BAP- Benzyl aminopurine; IAA-Indole-3-acetic acid; IBA-Indole-3-butyric acid; Kn- Kinetin; NAA- -Naphthalene acetic acid.

IntroductionMultiplication of plants in vitro through

plant tissue culture methods have emerged inlate 1980s as a valuable approach to increaseplant productivity (Gupta et al. 1993). In recentyears, there has been an increased interest in invitro culture techniques which offer a viabletool for mass multiplication and germplasmconservation of rare, endangered, aromatic andmedicinal plants (Arora and Bhojwani 1989;Sudha and Seeni 1994). Solanaceae familycomprises a number of plants widely known forthe presence of variety of natural products ofmedicinal significance mainly steroidal lac-tones, glycosides, alkaloids and flavonoids. So-lanum viarum (D.) is an ample branched, prick-ly shrub 1-2 meters tall at maturity. It is a mem-ber of economically significant familySolanaceae. The plant grown in India as a rich-est source of steroids (Mullahey et al. 1987),which yields an economically vital source ofglycoalkaloide solasodine which is present in

the fruit is a pioneer for the steroid disogenin(Budhavari 1989). This is used in the productionof steroid hormone for treating cancer, rheumat-ic arthritis, addisons disease and for providingcontraceptives (Srinivasan 2005). Since the higheconomical and pharmological values of sec-ondary metabolites, industries are intensely at-tracted in utilizing the plant tissue culture tech-nology for large scale production of these sub-stances (Misawa 1994.). The present investiga-tion was performed to determine the role of dif-ferent plant growth regulators on direct shootregeneration from in vitro derived nodal ex-plants of Solanum viarum.

Materials and methodsPlant material and surface sterilization

Nodes of axillary branches were excisedfrom the in vitro regenerated shoots of S. viarumwere used as explants as a prerequisite for ex-periments. Therefore for the explant source the

49Int. J. Med. Arom. Plants In vitro direct shoot organogenesis from nodal explants of Solanum viarum

Mahadev et al. http://[email protected]

seeds were collected from mature and driedfruits of healthy plants of S. viarum that are cul-tured in the herbal garden, Dravidian University,Kuppam, A. P., India. Matured seeds were firstwashed under running tap water for 30 mins. totake away any adherent fruit tissues and driedjuice which might serve as an agent for fungalcontagion. Then the seeds were rigorouslywashed with 0.4% bavistin (w/v) a fungicideunder sterile conditions for an hour and washedthrice with sterile distilled water. This was fol-lowed by surface sterilization with 5% Tween-20 (v/v) for 15 mins. and the seeds were steri-lized in 70% alcohol (v/v) for 2 mins. Then theseeds were washed with 0.1% HgCl2 (w/v) for 2mins. Finally the seeds were washed thrice withsterile distilled water to remove the traces ofHgCl2.

Culture medium and culture conditionsThe seeds were inoculated on MS medium

(Murashige and Skoog, 1962) containing 0.8%agar (w/v) supplemented with various concen-trations of cytokinins BAP and Kn and auxins(NAA, IAA and IBA) previous to autoclave, themedium was adjusted to the pH of 5.8 and steri-lized for 20 mins. at 121C for 15 lbs pressure.The culture room conditions maintained for invitro cultures were 26C 2C and 60-70% rela-tive humidity. Light intensity was 3000 lux witha photoperiod of 18 hrs day light and 6 hrs indark. The primary shoots formed in vitro wereseparated aseptically and cultured on MS medi-um supplemented with BAP (2.0 mg l-1).

Sub culturingAxillary bud explants (1.0 2.0 cm) were

excised from the in vitro regenerated shoots ofS. viarum were inoculated on MS medium con-taining 3.0% sucrose and gelled with 0.8% agarsupplemented with various concentrations ofcytokinins like BAP, Kn alone or in combina-tion with auxins such as NAA, IAA and IBAwere used for shoot proliferation. The pH of themedium was adjusted to 5.8 before gelling withagar and autoclaved for 20 mins. at 1210C for 15lbs pressure. The cultures were maintained byregular intervals of 21 days on fresh MS medi-um.

Data analysisVisual observations were recorded on the

frequency in terms of number of cultures re-sponding for axillary shoot proliferation, shootdevelopment, number of shoots per explant, av-erage length of the regenerated shoots, numberof roots per shoot and average root length.

Statistical analysisAll the experiments were conducted with a

minimum of 20 explants. All assays were re-peated at least three times. The experimentaldata were statistically analyzed by one-wayANOVA using the DMRT (Duncans MultipleRange Test) (P < 0.05) and were presented asthe mean standard error (SE).

Results and discussionEffect of cytokinins alone on direct shoot regen-eration from in vitro derived axillary bud ex-plants of S. viarum

Effect of two different cytokinins BAP andKn on direct shoot regeneration was examinedseparately. After two weeks of inoculation shootbud primordial was emerged along the cut por-tions of nodal explants, later they were devel-oped as shoots. In BAP (0.5-3.0 mg l-1) alonesupplemented MS medium shoot initiation wasobserved and the maximum mean number ofshoots (10.30.31) was observed at (2.0 mg l-1)(Figure 1a) and mean shoot length ranged from(2-4 cm) respectively. The optimum concentra-tion of BAP was found to be (2.0 mg l-1). Thenumber of multiple shoots were decreased withfurther increase in BAP concentration (> 3.0 mgl-1). In Kn (0.5-3.0 mg l-1) alone supplementedMS medium shoot bud initiation was observedand the mean number of shoots ranged from(4.0-8.0) with mean shoot length (3.0-5.0 cm).The number of shoots were less compare toBAP supplemented medium (Table 1). Directregeneration from nodal explant is another sub-stitute stepfor clonal propagation andgermplasm conservation is a well-establishedfactor. In vitro regeneration of plants dependson the interaction between endogenous growthsubstances and the artificial growth regulatorswhich may be added to the medium.The stimu-



lation of direct shoot regeneration depends onthe nature of the plant organ from which the ex-plant was derived and is extremely dependenton plant (George 1993a). Nodes contain axillarymeristem which can generate direct proliferationof shoots. Preexisting meristems are the base ofthe two pathways: direct shoot organogenesisand direct shoot proliferation. Meristems are

highly reactive cells and plants regenerated fromthem generally could show no variation in geno-typic and phenotypic characters (Saeed et al.1997). In the present study the cytokinin alonesupplemented medium multiple shoots becomevisible after 2-3 weeks of inoculation this waswell reported in Celastrus paniculatus (Lakshmiand Seeni 2001).

Table 1: Direct shoot organogenesis from nodal explants of in vitro raised S. viarum micro shoots.Plant growth regulators

(mg l-1)Regeneration

frequency(%)

Mean no. ofshoots/explant

Meanshoot length

(cm)BAP Kn NAA IBA IAA0.51.02.03.0-

-

-

-

1.02.03.0-

-

-

1.02.03.0-

-

-

1.02.0-

-

-

-

-

-

0.51.02.03.0-

-

-

1.02.03.0-

-

-

1.02.03.0-

-

1.02.0

-

-

-

-

-

-

-

-

0.50.50.50.50.50.5-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.50.50.50.50.50.5-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

0.50.50.50.5

758085807880838082908678838088989287908583779290

6.50.13c9.20.24i

10.3 0.31j7.50.22de4.20.51a7.30.37d8.60.23gh6.80.42c8.70.20h

12.20.31k9.30.28i7.20.14d10.60.36j5.80.43b

14.00.26m22.30.41q16.10.51p10.50.17j13.40.61l7.80.28ef10.30.18j8.20.21fg11.80.53k7.90.36ef

4.50.15fg3.60.32bc2.40.23a

3.90.14cde5.20.26i4.70.19gh4.00.25cde3.70.52cd5.30.41i3.20.28b4.60.35gh4.10.15def3.20.13b

5.00.12hi5.40.22ij3.80.25cd4.70.52gh8.20.14m6.30.31k7.20.26l

4.30.24efg5.80.42j4.50.28fg6.30.51k

Data represent treatment means SE followed by different letter (s) within column indicate signifi-cant differences according to ANOVA and DMRT test (P < 0.05).

Effect of BAP in combination with auxin on di-rect shoot regeneration from in vitro derivedaxillary bud explants

BAP, Kn in combination with auxins (NAAor IBA or IAA) on direct shoot regeneration wasexamined. BAP (0.5-3.0 mg l-1) in combinationwith NAA, IBA and IAA (0.5 mg l-1) were usedto produce direct shoot regeneration from theaxillary bud explants of in vitro regenerated S.viarum micro shoots. After two weeks of inocu-lation shoot bud primordial was emerged alongthe cut portions of nodular explants, later theywere developed as shoots.In BAP and NAAsupplemented medium the mean number of

shoots were ranged from (8.7-12.2) was ob-tained with shoot length (3.2-5.3 cm) respective-ly (Figure 1b). Whereas the lower concentrationof cytokinin BAP (1.0 mg l-1) and auxin IAA(0.5 mg l-1) favored the mean shoot number(10.30.18). With further increase in concentra-tion of BAP the shoot number decreased to(8.20.21). The highest number of shoots wasobtained in the combination of BAP (2.0 mg l-1)and IBA (0.5 mg l-1) with (22.30.41) shoots(Figure 1c) with a shoot length (3.80.25 cm)(Table 1) but maximum shoot length was ob-tained on Kn (1.0 mg l-1) in combination withIBA (0.5 mg l-1). The shoot number were de-creased with further increase in the BAP con-



centration (> 2.0 mg l-1). BAP preferred formultiple shoot induction and proliferation fromnodal explants than Kn in the present study. Ingeneral, BAP is the most effective growth regu-lator for stimulation and shoot proliferation.BAP mimics as an inhibitor agent and drivesbeside apical dominance of shoot induction and

shoot bud formation (Wang and Charle 1991).Advantage of BAP over any cytokinin in induc-tion and proliferation of shoots has been verywell standardized in Quercus rober (Puddephatet al. 1997) and Solanum nigrum (Sridhar andNaidu 2011).

Table 2: Root organogenesis of in vitro derived shoot lets of S. viarum supplemented with variousconcentrations of IAA, NAA and IBA using half strength MS medium.

Plant growth regulators (mg l-1) Regenerationfrequency (%)

Mean number ofroots/shoot

Mean root length(cm)NAA IBA IAA

0.20.40.60.81.0-

-

-

-

-

-

-

-

-

-

-

-

0.20.40.60.81.0-

-

-

-

-

-

-

-

-

-

-

-

0.20.40.60.81.0

838578656087929890857580857870

7.20.27b9.30.34d11.20.42f14.50.35h8.30.31c10.40.22e13.30.36g19.20.22j14.10.35h10.30.21e6.50.42a9.70.43d13.20.30g15.60.26i11.60.51f

4.50.26g4.80.41g3.70.52ef2.40.27a2.60.13ab7.00.12h4.60.32g2.30.27a3.50.15de3.20.18d

3.60.28def3.30.25de4.00.16f3.00.29bc2.40.18a

Data represent treatment means SE followed by different letter (s) within column indicate signifi-cant differences according to ANOVA and DMRT test (P < 0.05).

Effect of Kn in combination with auxin on directshoot regeneration from in vitro derived axillarybud explants

Direct shoot induction from the in vitro de-rived axillary bud explants of S. viarum was al-so observed on Kn alone and in combinationwith auxins like NAA, IBA and IAA but thenumber of shoot formation was less when com-pared with BAP. The optimal concentration fordirect shoot regeneration using Kinetin was (2.0mg l-1) in combination with IBA (0.5 mg l-1)produced (13.40.61) mean number of shoots.The maximum shoot length (8.20.14 cm) wasobserved at Kn (1.0 mg l-1) and IBA (0.5 mg l-1)(Table 1). In Kn and NAA supplemented medi-um the maximum shoot number (10.60.36)was obtained with a mean shoot length(3.20.13 cm) at Kn (2.0 mg l-1) and NAA (0.5

mg l-1). Whereas in Kn and IAA supplementedmedium the maximum shoot number(11.80.53) was obtained with a mean shootlength (4.50.28 cm) at Kn (1.0 mg l-1) and IAA(0.5 mg l-1). With further increase in the concen-tration of kinetin number of shoots were de-creased.

The combination of cytokinin and auxinseems to have a collaboration effect, ascytokinins enhance cell division, stimulate axil-lary bud and adventitious shoot proliferation andauxins regulate cell elongation, tissue swellingand shoot expansion. The effect of auxins andcytokinins on enhancing shoot regeneration hasbeen reported in several spices, such as Echina-cea purpurea (Koroch et al. 2002) and Catalpaovate (Lisowska and Wysokinska 2000).



Figure 1: Direct regeneration of shoots from nodal explants of in vitro raised microshoots of S. viarum.a) Shoot bud initiation from nodal explant after 7 days of inoculation on MS medium + BAP (2.0 mg l-1)b) Initiation of multiple shoots from nodal explant on MS medium + BAP (2.0 mg l-1) + NAA (0.5 mg l-

1)c) Multiplication of shoots from nodal explant on MS medium + BA (2.0 mg l-1) + IBA (0.5 mg l-1)d) Elongated multiple shoots regenerated from nodal explant on MS medium + BAP (3.0 mg l-1) + IBA

(0.5 mg l-1)e) Initiation of roots from the regenerated shoots in vitro on MS medium + (IBA 0.6 mg l-1)f) Plantlet showing elongated root systemg) Hardened plantlet in polycups containing soil and vermiculite in 1:1 ratioh) Plantlet in field conditions.



In the present study in vitro derived nodalexplants promoted shoot bud regeneration inpresence of the cytokinin BAP, Kn alone or incombination with auxins like NAA/IBA/IAAand resulted in maximum number of multipleshoot induction. Among the auxins tested(NAA, IBA and IAA) in combination withBAP, natural auxin IBA proved to be the betterfor providing maximum number of shoots.Similar response was studied earlier in Platanusacerifolia wild (Liu and Bao 2003). The nextbest auxin which performed well was NAA incombination with BAP. The effect of BAP andNAA on direct shoot regeneration was reportedin Gysophila paniculata (Zukar et al. 1997) andSpilanthes acmella (Saritha and Naidu 2008).IAA in combination with Kn also have showedthe effect on direct shoot regeneration. Theseresults are in line with the observations of Steviarebaudiana (Preethi et al. 2011) and Cajanuscajan (Misra 2002).

In vitro rootingThe in vitro developed shoots were excisedfrom clump of shoots and transferred on MSmedium supplemented with different concentra-tion of auxins such as NAA, IBA and IAA (0.2-1.0 mg l-1). The best rooting response was ob-served on IBA (0.6 mg l-1) supplemented medi-um, (Figure 1e) where maximum number ofroots (19.20.22) were obtained followed byIAA (0.8 mg l-1), NAA (0.8 mg l-1) supplement-ed medium where (15.60.26), (14.50.35)number of roots were induced respectively. Thenumber of roots decreased with further increasein auxin concentration (>0.8 mg l-1) (Table 2).The excised shoots cultured on the MS mediumcontaining different auxin concentrations forrooting in which the IBA performed well inproducing the maximum number of roots/shootwith higher regeneration frequency these reportswere similar with the study on Mentha piperita(Sujana and Naidu 2011). Rooted shootsshowed the maximum percentage of survival.Among the plant growth regulators tested, inmost of the combinations, the comeback of di-rect regeneration from nodal explants were re-sulted in prompting shoot regeneration directlyfrom the nodal explants without forming

callus. The excised nodes from in vitro derivedshoots were used as explants for further regen-eration as this is also true in Cuminum cyminum(Esmaeil Ebrahimie et al. 2007).

Acclimatization and hardeningWell-developed rooted plants were carefully

removed from culture tubes and washed to re-move the leftovers of agar (Figure 1f). Thesehealthy shoots were transferred to tray contain-ing autoclaved soil and vermiculate in 1:1 ratiofor acclimatization (Figure 1g). For a period of8-10 days the plants were kept in polythenemembrane. After that the surviving plants weretransferred to pots and allowed to grow undernursery shade conditions (Figure 1h). Finallythese acclimatized plants were planted in fieldconditions with maximum survivability.

Acclimatization of regenerated plants to theoutside environment is the last stage in planttissue culture and its achievement depends ondifferent factors as proposed by various re-searchers (George and Sherrington 1984b). Priorother investigation workers studied acclimatiza-tion of micropropagated plants in the greenhouse under field environments (Preece and Sut-ter 1991).

ConclusionIn the present study, regeneration of S.

viarum was greatly influenced by different plantgrowth regulators supplemented in the media. Itcan be concluded that based on readily availablenodal explants of S. viarum reproducible in vitrodirect shoot regeneration and proliferation, hasbeen described. The results obtained in the pre-sent study could be of enormous significance,which contribute to the development in themicropropagation of this economically im-portant medicinal plant on commercial scale tolight the current day demand.

Acknowledgements: The authors wish to ex-press their gratitude to the UGC BSR (Non-SAP) New Delhi, India, for supporting this re-search work by providing financial assistance inthe form of fellowship.



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