An efficient protocol for in vitro propagation of Solanum nigrum L. from nodal explants

Abstract

An efficient method for in vitro propagation of Solanum nigrum L has been developed. The nodal explants were cultured on MS medium supplemented with different concentrations of BAP and KIN. The two cytokinins tested, KIN was found to respond well in shoot multiplication and number of shoots from the nodal explants when compared to BAP. Large number of shoots was produced from all the concentrations of both the cytokinins. The highest frequency of 100% shoot induction was observed on MS medium supplemented with 10-15 µM KIN and BAP. The number of shoots produced on the basal medium supplemented with BAP ranged between 40 and 46 and 43 and 49 with KIN 30 days after inoculation. Excised shoots were transferred to rooting medium containing different concentrations of IBA and 2,4-D for root induction. The roots were initiated and well developed on a medium fortified with 10-15 µM of both the auxins. Of the two auxins tested, more number of roots were produced on the medium containing IBA. Maximum number of 47 roots per shoot were produced in 30 days on MS medium supplemented with 10 µM IBA. The well rooted plantlets were transplanted to the paper cup for hardening and the well established plants were transferred to the field for acclimatization.

Full text

Journal of Agricultural Technology 2011 Vol. 7(4):1063-1073
Available online http://www.ijat-aatsea.com
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An efficient protocol for in vitro propagation of Solanum nigrum
L. from nodal explants
Padmapriya, H.1, Karthikeyan, A.V.P2*, Jahir Hussain, G.2, Karthi, C. 2
and Velayutham, P.2
1Department of Biotechnology, PRIST University, Vallam, Thanjavur – India, 2Department of
Botany, Government Arts College (Autonomous), Karur 639 005 - India
Padmapriya, H., Karthikeyan, A.V.P, Jahir Hussain, G, Karthi, C. and Velayutham, P. (2011)
An efficient protocol for in vitro propagation of Solanum nigrum L. from nodal explants.
Journal of Agricultural Technology 7(4):1063-1073.
An efficient method for in vitro propagation of Solanum nigrum L has been developed. The
nodal explants were cultured on MS medium supplemented with different concentrations of
BAP and KIN. The two cytokinins tested, KIN was found to respond well in shoot
multiplication and number of shoots from the nodal explants when compared to BAP. Large
number of shoots was produced from all the concentrations of both the cytokinins. The highest
frequency of 100% shoot induction was observed on MS medium supplemented with 10-15 µM
KIN and BAP. The number of shoots produced on the basal medium supplemented with BAP
ranged between 40 and 46 and 43 and 49 with KIN 30 days after inoculation. Excised shoots
were transferred to rooting medium containing different concentrations of IBA and 2,4-D for
root induction. The roots were initiated and well developed on a medium fortified with 10-15
µM of both the auxins. Of the two auxins tested, more number of roots were produced on the
medium containing IBA. Maximum number of 47 roots per shoot were produced in 30 days on
MS medium supplemented with 10 µM IBA. The well rooted plantlets were transplanted to the
paper cup for hardening and the well established plants were transferred to the field for
acclimatization.
Key words: In vitro regeneration, shoot induction, rooting, hardening
Introduction
Solanum nigrum L. (Black nightshade) a member of Solanaceae is a
common herbaceous plant distributed everywhere. It is cultivated as a food
crop, both for its leaves and fruits. The stem may be glabrous or hairy. The
flowers usually white with five regular parts. The leaves alternate and some
what ovate with irregularly toothed wavy margin. The fruit is a round fleshy
berry and black when ripe. The seeds are brown and numerous (Gamble, 1921;
Edmonds and Chewya, 1997). The leaves and fruits contain toxic alkaloid
solanine. It is a glyco-alkaloid with the highest concentrations in the unripened
* Corresponding author: A.V.P. Karthikeyan, e-mail:avpkarthi@yahoo.com

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berries (Cooper and Johnson, 1984). When ripe, the berries are the least toxic
part of the plant and are sometimes eaten without ill effects (Watt and Breyer-
Brandwijk, 1962). Solanine may be separated by chromatography into six
components (Merck, 1989). Solanidine is obtained after hydrolysis of solanine,
and is less toxic. The leaves and fruits are popularly used as vegetable in the
preparation of soup. The leaves blend well with other greens and pulses to
make porridges in southern parts of India. Besides being used for human
consumption, the leaves serve as fodder and browse for domestic herbivorous
animals (Akubugwo, et al, 2007). The whole plant is antiperiodic,
antiphlogistic, diaphoretic, diuretic, emollient, febrifuge, narcotic, purgative
and sedative (Singh and Kachroo, 1976; Emboden, 1979; Lust, 1983; Duke and
Ayensu, 1985). It is harvested in the autumn when both flowers and fruit are
upon the plant, and is dried for later use (Grieve, 1984). The leaves, stems and
roots are used externally as a poultice, wash, etc. in the treatment of cancerous
sores, boils, leucoderma and wounds (Duke and Ayensu, 1985; Moerman,
1998). Extracts of the plant are analgesic, antispasmodic, anti-inflammatory and
vasodilator (Duke and Ayensu, 1985; Ravi et al, 2009). The plant has been used
in the manufacture of locally analgesic ointments and the juice of the fruit has
been used as an analgesic for toothaches (Chiej, 1984). The plant has also been
used as insecticide (Merck, 1989) and larvicide (Singh, et al., 2001).
Micropropagation is the practice of rapidly multiplying stock plant
material to produce a large number of progeny plants, using modern plant tissue
culture methods. It is a method of vegetative propagation conducted in the
laboratory condition and it has a significant impact on plant breeding,
horticulture and medicine. It is the ever-ready tool for specialization in
hybridization either by sexual or asexual means. It is a suitable method for
obtaining a large quantity of genetically homogenous and healthy plant material
which can be used for planting (Pierik, 1987). This technique is an alternative
method of propagation as there is an increase in the propagation rate of plants,
availability of plants throughout the year, protection of plants against pests and
pathogens under controlled conditions and the availability of uniform clones
and uniform production of secondary metabolites (Bajaj, et al.,1988). Some
micropropagation works have been conducted from the various explants of S.
nigrum. Akhterjahan and Hadiuzzaman (1996) obtained plant regeneration
from shoot tip, stem, leaf and root segments of S. nigrum through callus culture.
They obtained callus on MS medium with NAA, regeneration of shoots on
BAP and rooting on IBA. Hassanein and Soltan (2000) cultured shoots from
shoot cutting of germinated seeds of S. nigrum on different media (B5, MS or
SH) and observed the best culture condition for shoot formation was the culture
of stem internode segments on B5 medium supplemented with 0.5 mg dm-3

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BAP at 16-h photoperiod (irradiance of 100 µmol m-2 s-1). Direct
organogenesis and in vitro flowering was obtained in S. nigrum by Venugopal,
et al. (2005). The highest frequency and number of multiple shoots were
obtained from leaf and nodal explants on MS medium supplemented with
benzyladenine and IAA. Regenerated plants rooted and flowered on rooting
medium supplemented with IBA or IAA. Basha, et al. (2008) made a successful
induction of callus from S. nigrum L. on MS basal medium supplemented with
IAA and BAP. Regeneration shoots from callus and in vitro flowering were
obtained on MS medium fortified with BAP and IAA or NAA or 2,4-D. The
best rooting was obtained on MS containing 0.5 mg/l IBA. Kannan et al. (2005)
reported the in vitro regeneration of S. nigrum using different plant growth
regulators and concluded that BAP 0.5 mg/l, 2, 4-D 1.0 mg/l and IBA gave the
highest frequency of the well growing shoot. Yogananth, et al (2009) showed
the accumulation of the alkaloid solasodine in the callus of S. nigrum. Hanan et
al. (2010) produced S. nigrum with a high power of alkaloid accumulation
through in vitro regeneration trials followed by in vivo plant acclimatization.
MS-basal medium containing BA and NAA (0.5 mg/ml each) was the best for
both plants. A series of in vitro and in vivo plants were successfully produced
and chemical analysis revealed contents of glycoalkaloids higher than those
reported for intact field plants. Bhat et al. (2010) obtained high frequency of
shoots directly from the leaf explant of S. nigrum on MS medium supplemented
with BAP and KIN without any callusing stage. Though some
micropropagation studies have been conducted so far, this paper deals with the
efficient plant regeneration system with large number of shoots within a short
period.
Materials and Methods
Sterilization, Media and Explants
All the glassware was washed thoroughly with chromic acid (potassium
dichromate and sulphuric acid, 2:1 w/v), rinsed in tap water and then with
distilled water. Sterilization of glassware, forceps and scalpels for
micropropagation was done in an autoclave at 121°C for 20 minutes at 1.06 kg
cm–2. MS basal medium (Musarhige and Skoog, 1962) was used along with
different concentrations of Plant Growth Regulators for shoot multiplication
(BAP and KIN) and rooting (IBA, NAA and 2,4-D). The pH of the medium
was adjusted to 5.8 with 0.1 N NaOH or 1 N HCl prior to autoclaving (121°C at

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1.06 kg cm–2 for 20 min). The explants were collected from the field grown
plants of Solanum nigrum L. in and around the college campus. The plant
materials, nodal explants with axillary bud, were rinsed with running tap water
with few drops of liquid soap (Teepol). The explants were then washed with
distilled water 3 or 4 times and further sterilization was carried out in Laminar
Air Flow chamber under aseptic condition prior to inoculation. The explants
were sterilized with 70% alcohol for 30-45 sec and 0.1% (w/v) HgCl2 for 5
min. The explants were then washed 4-5 times with sterile distilled water.
Inoculation Procedure
For multiple shoot induction, the nodal explants with axillary bud were
placed on MS medium supplemented with different concentrations of
benzylaminopurine (BAP: 5-25 µM) or kinetin (KIN: 5-25 µM). In vitro raised
shoots of 2 cm and above were excised from the culture tube and subcultured
into MS medium supplemented with various concentrations (2-10 µM) of
indole butyric acid (IBA: 5-25 µM) or 2,4-dichlorophenoxy acetic acid (2,4-D:
5-25 µM). The root number and length were measured in each culture medium.
Culture maintenance and conditions
All cultures were maintained at 25±2°C in a culture room under cool
white fluorescent lamps (Phillips, India) at intensity of 50 µmol m–2 s–1 with
16 hrs photoperiod.
Hardening and Acclimatization
Plantlets with well-developed roots were dislodged from the culture
medium and roots were washed gently under running tap water to remove the
adhering medium. Plantlets were transferred to plastic cups (10 cm diameter)
containing autoclaved garden soil, farmyard manure and sand (2:1:1). Each
plantlet was irrigated with distilled water every 2 days for 2 weeks followed by
tap water for one week. The potted plantlets were initially maintained under
culture room conditions in 3 weeks and later transferred to normal laboratory
conditions in 2 weeks. The potted plantlets were initially covered with porous
polyethylene sheets to maintain high humidity and were maintained inside the
culture room. The relative humidity was reduced gradually. After 30 days the
plantlets were transplanted to the field under shade for 3 weeks and then
transplanted to the soil for further growth and development.

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Experimental Design, Data Collection and Statistical Analysis
All experiments were preformed using a randomized comletely block
design and each experiment consisted of five explants per flask and five
replicate culture flasks per plant growth regulator treatment. The parameters
recorded were frequency (number of cultures responding in terms of multiple
shoot proliferation and root development), number of shoots per explant, shoot
length, number of roots per shoot, root length and survival rate (%). All of the
experiments were repeated five times. The analysis of variance (ANOVA)
appropriate for the design was carried out to detect the significance of
differences among the treatment means were compared using Duncan’s
Multiple Range Test (DMRT) at a 5% level of significance (Gomez and
Gomez, 1976).
Results and Discussion
Shoot Multiplication
Shoot multiplication was observed on MS medium supplemented with all
the concentrations of BAP and KIN within 10 days of inoculation (Fig. 1a).
Shoot multiplication frequency was observed on both the hormones (Fig. 1b,c).
The percentage of shoot induction frequency ranged from 80-100% and 90-
100% on MS medium supplemented with BA and KIN respectively (Table 1).
Large number of shoots was produced in 30 days of culture. The highest
frequency of 100% shoot induction was observed on MS medium supplemented
with 10-15 µM KIN and BAP.
However, variations among the two hormones were observed in number
of shoots and shoot length. The number of shoots produced on the basal
medium supplemented with BAP ranged between 40 and 46 with the highest in
15 µM BAP 30 days after inoculation. The number of shoots ranged between
43 and 49 on MS medium supplemented with KIN with the highest in 10-15
µM KIN (Table 1) in the same period of culture. Of the two cytokinins tested,
KIN was found to respond well in shoot multiplication and number of shoots
from the nodal explants when compared to BAP. A large number of shoots was
formed from the nodal explants of S. nigrum within a short period of 45-60
days.

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The shoot length varied from 7-9 cm in both the cytokinins. Shoots
growing on MS medium supplemented with 15 µM BAP reached 9.24 cm
height in 30 days of culture. Shoots growing on MS medium with 15 µM KIN
reached 9.46 cm height in the same period (Table 1).These results showed both
the cytokinins tested were found to initiate and proliferate shoots from the
nodal explants of S. nigrum. However, KIN was found to be more suitable for
shoot multiplication when compared to BAP.
The shoot induction and proliferation depend on plant growth regulators
and types of explants (Patnaik and Chand, 1996; Mohamed et al., 1999). In
many plants, multiple shoots were obtained from the shoot tips or axillary buds
by administering BAP or KIN (Kumar et al., 1998; Sahoo and Chand, 1998;
Velayutham, 2003; Baskaran and Jayabalan, 2005; Bhat et al., 2010). In the
present study, a large number of shoots was produced from the nodal explant of
S. nigrum on MS medium supplemented with both BAP and KIN separately
within a short period of 30-45 days.

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Table 1. Effect of different concentrations of cytokinins on shoot induction from the nodal
explant of Solanum nigrum L.
BAP KIN Percentage of response Number of shoots Shoots lenght
5 µM - 95
b
41 ± 0.96 c 7.26 ± 1.86 de
10 µM - 95
b
43 ± 1.24 b 8.68 ± 2.00 b
15 µM - 100
a
47 ± 1.86 b 9.24 ± 1.96 a
20 µM - 95
b
46 ± 1.56 a 7.58 ± 1.96 c
25µM - 85
c
40 ± 1.96 cd 7.48 ± 1.86 d
- 5 µM 90
c
40 ± 1.33 cd 8.65 ± 1.32 de
- 10 µM 100
a
45 ± 0.96 c 9.42 ± 1.96 ab
- 15 µM 100
a
49 ± 1.32 a 9.46 ± 1.86 a
- 20 µM 95
b
43 ± 0.96 d 9.12 ± 1.95 c
- 25 µM 95
b
43 ± 0.96 d 8.84 ± 1.96 d
Values in the last two columns are Mean ± SE of Mean followed by the letters within the
column indicating the level of significance at P<0.05 by Duncan’s Multiple Range Test (same
letter within the column of the respective growth regulator indicates the absence of difference;
different letters indicate the significant difference; and combination of letters indicate no
significant difference)
Though several growth regulators are available for shoot multiplication,
BAP and KIN are widely used. Of the two cytokinins tested, KIN was more
effective in shoot induction and proliferation than BAP. In several studies BAP
was more effective in inducing bud break resulting in the sprouting of a large
number of shoots (Sahoo and Chand, 1998; Kadota and Niimi, 2003;
Velayutham and Ranjithakumari, 2003; Martinussen et al., 2004; Vasudevan et
al., 2004). However, in the present investigation, KIN was found to be more
efficient in shoot multiplication.
Rooting
Excised shoots were transferred to rooting medium containing different
concentrations of IBA and 2,4-D (5-25 µM) for root induction. Root initiation
was observed on MS medium supplemented with all concentrations of IBA and
2,4-D in 10 days of culture (Fig.1d,e). The root induction frequency was more

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or less similar in all the concentrations of both the auxins (Table 2). However,
the roots were initiated and well developed on a medium fortified with 10-15
µM of both the auxins (Fig.1d,e; Table 2). Of the two auxins tested, more
number of roots were produced on the medium containing IBA. Maximum
number of 47 roots per shoot with the mean length of 5.5 cm were produced in
30 days on MS medium supplemented with 10 µM IBA (Table 2).
Table 2. Effect of different concentrations of auxins on root induction from the isolated
shoots of Solanum nigrum L. (after 30 days)
BAP KIN Percentage of response Number of shoots Shoots lenght
5 µM - 95 42± 1.96 bc 4.73± 2.01 bc
10 µM - 95 47± 0.95 a 5.50± 0.75 a
15 µM - 100 45± 1.20 ab 5.00± 1.96 b
20 µM - 95 35± 2.66 d 4.65± 2.93 cd
25µM - 85 30± 2.98 e 3.90± 2.26 e
- 5 µM 85 27± 2.91 d 3.80± 2.72 cd
- 10 µM 95 33± 2.36 b 4.45± 1.82 b
- 15 µM 100 36± 1.20 a 4.95± 0.96 a
- 20 µM 90 30± 2.52 c 3.90± 2.12 c
- 25 µM 80 25± 2.88 de 3.65± 2.72 de
Values in the last two columns are Mean ± SE of Mean followed by the letters within the
column indicating the level of significance at P<0.05 by Duncan’s Multiple Range Test (same
letter within the column of the respective growth regulator indicates the absence of difference;
different letters indicate the significant difference; and combination of letters indicate no
significant difference
In most of the studies IAA, IBA and NAA were used for root induction.
High frequency of rooting was achieved by IAA in Syzygium cuminii (Jain and
Babbar, 2003), Gossypium arboreum and G. hirsutum (Bajaj and Gill, 1986)
and IBA in Aristolochia indica (Manjula et al., 1997), Gymnema sylvestris
(Komalavalli and Rao, 2000), Avicennia marina (Al-Bahrany and Al-Khayri,
2003) and Eclipta alba (Baskaran and Jayabalan, 2005). Higher frequency of
roots was observed in Cichorium intybus at 5 µM NAA (Velayutham and
Ranjithakumari, 2003), Rubus chamoemorus (Martinussen et al., 2004),
Viburnum odoratissimum (Schoene and Yeager, 2005), Plumbago zeylanica at

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3 µM NAA (Velayutham et al., 2005) and Aerva lanata at 6 µM NAA
(Suganya et al., 2005). Jabeen et al. (2005) also showed that NAA was found to
induce more number of roots when compared to IAA and IBA in Solanum
nigrum. In the present study also IBA was found to induce more number of
roots when compared to 2, 4-D.
Hardening and acclimatization
The well rooted plantlets were transplanted to the paper cup (Fig. 1f)
containing a mixture of autoclaved red soil, farm yard manure and sand in the
ratio of 2:1:1. The survival rate of these plants was 80%. The established plants
were transferred to the field for acclimatization.
References
Akhter jahan, M. A. and Hadiuzzaman, S. (1996). Callus Induction and Plant Regeneration
from Different Explants of Solanum nigrum L. Seedlings. Plant Tissue Cult. 6(1): 57-
62.
Akubugwo, I.E., Obasi, A.N., and Ginika, S.C. (2007). Nutritional Potential of the Leaves and
Seeds of Black Nightshade-Solanum nigrum L. Var virginicum from Afikpo-Nigeria.
Pakistan Journal of Nutrition 6(4): 323-326.
Al-Bahrany, A .M. and Al-Khayri J. M. (2003). Micropropagation of grey mangrove Avicennia
marina. Plant Cell, Tissue and Organ Culture 72: 87-93.
Bajaj, Y.P.S. and Gill, M.S., (1986). Micropropagation and Germplasm. Preservation of cotton
(Gossypium spp.) through shoot tip and meristem cultufre. Ind. J. Exp. Biol. 24: 581-
583.
Bajaj,Y.P.S., Furmanowa, M. and Olszowsks, O., (1988). Biotechnology of the
micropropagation of medicinal and aromatic plants. In: Biotechnology in Agriculture
and Forestry, Medicinal and aromatic Plants I. (ed.) Bajaj, Y.P.S.). Springer - Berlin,
Heidelberg, New York, Tokyo, 4 : 60-103.
Basha, Amzad Kolar, L .Vivekanandan and Ghouse Basha M. (2008). In vitro Regeneration
and Flower Induction on Solanum nigrum L. from Pachamalai hills of Eastern Ghats.
Plant Tissue Cult. & Biotech. 18(1): 43-48.
Baskaran, P. and Jayabalan, N. (2005). An efficient micropropagation system for Eclipta alba -
a valuable medicinal herb. In Vitro Cell. Dev. Biol.Plant 41:532-539.
Bhat, M.A., Mujib, A., Junaid, A. and Mohamooduafar, M. (2010). In vitro regeneration of
Solanum nigrum with enhanced solasodine production. Biologia Plantarum 54(4) 757-
760.
Chiej, R. Encyclopaedia of Medicinal Plants. MacDonald 1984 ISBN 0-356-10541-5Cooper
MR; Johnson AW, (1984). Poisonous Plants in Britain and other effects on Animals
and Man. Ministry of Agriculture, Fisheries and Food, Ref. Book no. 161, London pp
219-220.
Duke, J. A. and Ayensu. E. S. (1985). Medicinal Plants of China Reference Publications, Inc.
ISBN 0-917256-20-4

Journal of Agricultural Technology 2011 Vol. 7(4):1063-1073
1072
Edmonds, J.M. and J.A. Chweya. (1997). Black nightshades Solanum nigrum L. and related
species. Institute of Plant Genetics and Crop Plant Research/ International Plant
Genetic Resources Institute, Rome.
Emboden, W. Narcotic Plants Studio Vista 1979 ISBN 0-289-70864-8.Gamble, J.S. (1921).The
Flora of Presidency of Madras. Adlard & Son Limited, London. 2
Gomez, K. A. and Gomez, K. A. (1976). Statistical procedures for agricultural research with
emphasis on Rice. Philippines International Rice Research Institute, Los Bans.Grieve,
A Modern Herbal. Penguin.
Hanan Abd, Al-Hay and Al-Ashaal, A, (2010). Regeneration, in vitro glycoalkaloids production
and evaluation of bioactivity of callus methanolic extract of Solanum tuberosum L.
Fitoterapia 81( 6) 600-606
Hassanein, A.M. and Soltan, D.M. (2000). Solanum nigrum is a Model System in Plant Tissue
and Protoplast Cultures. Biologia Plantarum 43(4) :501-509.
Hassanein, A.M., Ahmed, A.M., Abed-El-Hafez, A.I.I., Soltan, D.M. (1999). Isoenzyme
Expression During Root and Shoot Formation in Solanum nigrum. Biologia Plantarum
42(3) 341-347.
Jabeen, F.T.Z., Venugopal, R.B., Kiran, G., Kaviraj, C.P. and Rao, S. (2005). Plant
regeneration and in vitro flowering from leaf and nodal explants of Solanum nigrum
(L). - An important medicinal plant. Plant Cell Biotechnology and Molecular Biology
6(1&2):17-22.
Jain, N. and Babbar, S.B. (2003). Regeneration of 'juvenile' plants of black plum, Syzygium
cuminii Skeels, from nodal exp of mature trees. Plant Cell, Tissue and Organ Culture
73: 257-263.
Kadota, M. and Niimi, Y. (2003). Effects of cytokinin types and their concentrations on shoot
proliferation and hyperhydricity in in vitro pear cultivar shoots. Plant Cell, Tissue and
Organ Culture 72: 261-265.
Kannan, T.M.S., S.M. Nagarajan and S. Kulothungan, (2005). Micropropagation of Solanum
nigrum L. a medicinal herb. Plant Archieves: 609 305.
Komalavalli, N. and Rao, M.V., (2000). In vitro micropropagation of Gymnema sylvestre - A
multipurpose medicinal plant. Plant Cell Tiss. Org. Cult. 61:97-105.
Kumar, S., Chander, S., Gupta, H. and Sharma, D.R. (1998). Micropropagation of Actinidia
deliciosa from axillary buds. Phytomorphology 48(3): 303-307.
Lust. J. 1983. The Herb Book. Bantam books. ISBN 0-553-23827-2.
Manjula, S., Anita, T., Benny, D. and Nair, C.M., (1997). In vitro plant regeneration of
Aristolachia indica through axillary shoot multiplication and organogenesis. Plant Cell
Tiss. Org. Cult. 51: 145-148.
Martinussen, I., Nilsen, G., Svenson, L., Junttila, O. and Rapp, K.(2004). In vitro propagation
of cloudberry (Rubus chamaemorus). Plant Cell, Tissue and Organ Culture 78: 43-
49.Merck Index. 1989 Ed. S. Budavari, 11th Ed., NJ, USA. pp:1371.
Moerman, D. (1998). Native American Ethnobotany Timber Press. Oregon. ISBN 0-88192-
453-9
Mohamed, S.V., Jawahar, M., Thiruvengadam, M., Jeyakumar, M. and Jayabalan, N. (1999).
Effect of cytokinins on the proliferation of multiple shoots in horse gram
(Macrotyloma uniflorum (L.) Verdc.). Jour. Plant Biotech.1: 79-83.
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassay with
tobacco tissue culture. Physiologia Plant., 15, 473-497.
Patnaik, J. and Chand, P.K. (1996). Micropropagation of Hemidesmus indicus (L.) R. Br.
through axillary bud culture. Plant Cell Rep.15: 427-430.

Journal of Agricultural Technology 2011 Vol. 7(4):1063-1073
Available online http://www.ijat-aatsea.com
ISSN 1686-9141
1073
Pierik, R.L.M. (1987). In vitro Culture of Higher Plants. Martinus Nijhoff Publishers. The
Hague. Plant Tissue Culture. 14 (1): 9-16.
Ravi, V., Saleem, T.S.M., Patel, S.S., Raamamurthy, J. and Gauthaman, K. (2009). Anti-
Inflammatory Effect of Methanolic Extract of Solanum nigrum Linn Berries.
International Journal of Applied Research in Natural Products 2(2): 33-36.
Sahoo, Y. and Chand, P.K., 1998. In vitro multiplication of a medicinal herb, Tridax
procumbens L. (Mexican daisy, coat buttons) : Influence of explanting season, growth
regulator synergy, culture passage and planting substrate. Phytomorphology 48(2):
195-205.
Schoene, G. and Yeager, T. (2005). Micropropagation of sweet viburnum (Viburnum
odoratissimum). Plant Cell, Tissue and Organ Culture 83: 271-277.
Singh, Dr. G. and Kachroo. (1976). Prof. Dr. P. Forest Flora of Srinagar. Bishen Singh
Mahendra Pal Singh.
Singh, S.P., Raghavendra, K., Singh, R.K. and Subbarao, S.K. (2001). Studies on larvicidal
properties of leaf extract of Solanum nigrum Linn. (family Solanaceae). Cur. Sci.
81(12): 1529-1530.
Vasudevan, A., Selvaraj, N., Ganapathi, S., Kasthurirengan, S., Ramesh Angbazhagan and
Manickavasagam, M. (2004). Glutamine: a suitable nitrogen source for enhanced
shoot multiplication in Cucumis sativus L. Biologia Plantarum 48(1): 125-128.
Velayutham, P. (2003). In vitro Regeneration, Pharmacognosy and Antimicrobial Activities of
Cichorium intybus L. Ph.D. Thesis submitted to Bharathidasan University,
Tiruchirappall.
Velayutham, P. and Ranjitha Kumari, B.D. (2003). Direct shoot regeneration from leaf explants
of chicory (Cichorium intybus L.). Plant Cell Biotech Mol Bio. 4(3&4): 125-130.
Velayutham, P., Jahir Hussain, G. and Baskaran, P. (2005). In vitro plantlet formation from
nodal explants of Plumbago zeylanica L. - an important medicinal plant. J. Swamy
Bot. Cl. 22 : 117-120.
Velayutham, P., Ranjithakumari, B.D., and Baskaran, P. (2006). An effeicient in vitro plant
regeneration system for Cichorium intybus L. an important medicinal plant. J.
Agricultural Technology. 2(2):287-298.
Venugopal, R. B., Kaviraj, C. P., Srinath Rao, Jabeen, F. T. Z., Kiran, G. (2005). Plant
regeneration and in vitro flowering from leaf and naodal explants of Solanum nigrum
(L.) - an important medicinal plant. Plant Cell Biotechnology and Molecular Biology,
6 (1/2): 17-22.
Watt, J. M. and Breyer-Brandwijk, M.G. (1962). The Medicinal and Poisonous Plants of
Southern and Eastern Africa E & S, Livingston Ltd., Edinburgh and London, UK, pp
996-1000.
Yoganath, N., Bhakyaraj, R., Chanthuru, A., Parvathi, S. and Palanivel, S. (2009). Comparative
Analysis of Solasodine from in vitro and in vivo cultures of Solanum nigrum Linn.
Kathmandu University Journal of Science. Engineering and Technology 5(1) 99-103.
(Received 9 January 2011; accepted 15 May 2011)