Applications in plant tissue culture

 (1) Extracts from black carrot tissue culture as
potent anticancer agents.
 (2) Enhanced production of tropane alkaloids in
transgenic Scopolia parviflora hairy root cultures
over-expressing putrescine N-methyl transferase
(PMT) and hyoscyamine-6β-hydroxylase (H6H).
 (3) Taxus globosa S. cell lines: initiation,
selection and characterization in terms of
growth, and of baccatin III and paclitaxel
production.
 (4) Production of camptothecin in cultures of
Chonemorpha grandiflora.

 APPLICATION IN PLANT TISSUE CULTURE (CONT.,)
 (5) Regeneration, in vitro glycoalkaloids
production and evaluation of bioactivity of callus
methanolic extract of Solanum tuberosum L.
 (6) The influence of medium composition on
alkaloid biosynthesis by Penicillium citrinum.
 (7) Differential production of tropane alkaloids in
hairy roots and in vitro cultured two accessions
of Atropa belladonna L. under nitrate treatments.
 (8) Increased vincristine production from
Agrobacterium tumefaciens C58 induced shooty
teratomas of Catharanthus roseus G. Don.

 (9) Enhancement of taxane production in hairy
root culture of Taxus x media var. Hicksii
 (10) An endophytic taxol-producing fungus from
Taxus media, Cladosporium cladosporioides MD2.
 (11) Optimized nutrient medium for galanthamine
production in Leucojum aestivum L. in vitro shoot
system.
 (12) Salinity stress enhances production of
solasodine in Solanum nigrum L.

 (13) Biosynthesis of hyperforin and
adhyperforin from amino acid precursors in
shoot cultures of Hypericum perforatum.

Plant Foods Hum Nutr. 2013 Sep

 Black carrots contain anthocyanins.
Explants of young black carrot shoots were
cultured in Murashige and Skoog (MS)
medium for callus initiation and were
transferred to new MS medium
supplemented with four different
combinations of 2,4-dichlorophenoxyacetic
acid and kinetin.
 Subsequently, the lyophilized calli and
black carrot harvested from fields were
subjected to ultrasound extraction with
ethanol at a ratio of 1:15 (w:v).

 Extracts applied to
(human breast adenocarcinomas), (human
colon adenocarcinoma), (human
prostate adenocarcinoma), Neuro 2A
(Musmusculus neuroblastoma) cancer cell lines
and VERO (African green monkey kidney) normal
cell line by MTT assay.
 The highest cytotoxic activity was achieved
against Neuro-2A cell lines exhibiting viability of
38-46% at 6.25 μg/ml concentration for all calli
and natural extracts. However, a significantly
high IC50 value of 170.13 μg/ml was attained in
normal cell line VERO indicating that its natural
counterpart is an ideal candidate for treatment
of brain cancer without causing negative effects
to normal healthy cells.

•In Vitro Cellular & Developmental Biology Plant . Aug2011, Vol. 47 Issue 4, p516-
524. 9p.
scopolamine
Scopolia parviflora

 Scopolia parviflora adventitious roots were
metabolically engineered by co-expression of
the two gene putrescine N-methyl
transferase (PMT) and hyoscyamine-6β-
hydroxylase (H6H) cDNAs with the aid of
Agrobacterium rhizogenes.
 The transformed roots developed into
morphologically distinct S. parviflora PMT1
(Sp PMT1), S. parviflora PMT2 (Sp PMT2), and
S. parviflora H6H (Sp H6H) transgenic hairy
root lines.
 Consequent to the introduction of these key
enzyme genes, the production of the
alkaloids hyoscyamine and scopolamine was
enhanced.

 Among the transgenic hairy root lines, Sp
PMT2 line possessed the highest growth
index.
 The treatment of transgenic hairy roots
with growth regulators further enhanced
the production of scopolamine.
 Thus, the results suggest that PMT1, PMT
2, and H6H genes may not only be involved
in the metabolic regulation of alkaloid
production but also that these genes may
play a role in the root development.

Baccatin III
Biocell. 2010 Apr;34(1):1-6.
Taxus globosa

 Of the initial six cell lines originating from
explants of Taxus globosa, or Mexican yew
(stem internode,leaves,meristematic tissue)
 Three were selected for their microbial and
oxidation resistance, two from leaves and
the other from stem internode
 A study of their behavior, both in terms of
cell growth, and of baccatin III and paclitaxel
production, was developed in suspension
cultures with an initially standardized
biomass (fresh weight 0.23 g/L) using
modified Gamborg's B5 medium, and an
elicitor (methyl jasmonate), on either the
first or seventh day of culture, at several
levels (0, 0.1, 1, 10, 100 microM).

 In most of the conditions used, the three
cell lines showed growth associated
baccatin III production.
 The cell line from stem internode was the
highest producer of baccatin III using 1
microM elicitor, sampling at 10 days (p < or
= 0.01, 6.45 mg/L).
 This same line also had the highest biomass
production (6.85 g/L, p < or = 0.01) at 10
days of culture but at the higher elicitor
concentration of 10 microM.
 All three cell lines did not produce
paclitaxel under experimental conditions
used.

camptothecin
Pharmacognosy Res. 2010 Sep;2(5):296-9. doi: 10.4103/0974-8490.72327.
Chonemorpha grandiflora

 METHODS:
 Callus cultures of C. grandiflora were
raised on Murashige and Skoog's medium
supplemented with 2, 4-D.
 Stem with bark and callus were used for
phytochemical analysis mainly the
alkaloids.
 Detection and identification of
camptothecin was carried out using
thin-layer chromatography (TLC), high-
performance thin-layer chromatography,
(HPTLC) and high-performance liquid
chromatography (HPLC).

 RESULTS:
 An important anticancer alkaloid,
camptothecin was detected in ethanolic
extracts of stem with bark and callus
cultures of C. grandiflora. camptothecin
content was 0.013 mg/g in stem with bark
and 0.003 mg/g in callus.
 CONCLUSION:
 This is the first report on in vivo and in vitro
production of camptothecin in C. grandiflora.
Camptothecin is known to occur only in
six plant sources so, alternative sources for
camptothecin are needed. Thus of C.
grandiflora could be anew promising
alternative source of camptothecin

Glycoalkaloid
Fitoterapia. 2010 Sep;81(6):600-6. doi: 10.1016/j.fitote.2010.03.001. Epub 2010 Mar 20.
Solanum tuberosum

 Callus and differentiated shoots initiated
from Solanum tuberosum L. on MS media
containing BA, IAA, and Kin. Glycoalkaloids
are produced in callus and shoots in
concentrations higher than original tubers
using HPLC.
 Callus methanolic extract had promising
anticancer activity with low IC(50) values
against human carcinoma cell lines of
breast, lymphoplastic leukemia, larynx,
liver, cervix, colon, and brain.
 IC(50) (microg/mL) were 2.7, 3.7, 6, 6.7,
10, 13.6, and 22.3 respectively.

 Antioxidant capacity of the extract (76.4%)
performed using ESR. Preliminary screening
showed that the extract exhibited in vitro
virucidal activity against Herpes simplex.
 The extract possessed in vitro
schistomicidal and fasciolicidal activity.

clavine ergot alkaloids
quinoline alkaloids
Prikl Biokhim Mikrobiol. 2010 Sep-Oct;46(5):572-6.

 The fungus P. citrinum produces secondary
metabolites, clavine ergot alkaloids (EA), and
quinoline alkaloids quinocitrinines (QA) in
medium with various carbon and nitrogen
sources and in the presence of iron, copper,
and zinc additives.
 Mannitol and sucrose are most favorable for
EA biosynthesis and mannitol is most
favorable for QA.
 Maximum alkaloid production is observed on
urea.

 Iron and copper additives in the medium
containing zinc ions stimulated fungal growth
but inhibited alkaloid biosynthesis.
 The production of these secondary
metabolites does not depend on the
physiological state of culture, probably due
to the constitutive nature of the enzymes
involved in biosynthesis of these substances

Z Naturforsch C. 2010 May-Jun;65(5-6):373-9.
Atropa belladonna

 Effects of nitrate concentrations (KNO3) on
the production of two tropane alkaloids,
hyoscyamine and scopolamine.
 The growth of aerial parts and roots of two
in vitro propagated accessions of Atropa
belladonna and hairy roots were studied .
 As hairy roots cultures are able to keep a
stable production of alkaloids over long
periods of subculturing, they are considered
as an interesting option for the study of
alkaloid biosynthesis.
 A hairy roots culture of Atropa belladonna
was established by transformation with
Agrobacterium rhizogenes strain AR15834.

 The results of our study showed that a rise in
KNO3 concentration caused a decline in hairy
roots growth, and had a remarkable effect on
the alkaloid content.
 The alkaloid concentrations obtained in the
hairy roots were 3-20 times higher than that
in the plants at 35 mM of KNO3.
 Increasing the nitrate concentration in the
medium of hairy roots also improved the
hyoscyamine/scopolamine ratio, while it
increased the scopolamine/hyoscyamine
ratio in the studied plants

vincristine
Nat Prod Res. 2009;23(11):973-81. doi: 10.1080/14786410802131153.
Catharanthus roseus

 Dimeric indole alkaloids are used extensively
for cancer therapy.
 Agrobacterium tumefaciens C58 strain was
used for induction of shooty teratoma in
Catharanthus roseus using epicotyl and stem
node explants.
 The transformed nature of shooty teratomas
was confirmed by nopaline assay.
 Growth kinetics of shooty teratomas depicted
maximum growth during 21-24 days of culture.
 Dimeric alkaloid vincristine in the transformed
cultures was present at a concentration of
0.011 that was tenfold higher compared to
untransformed control cultures

Baccatin III
J Plant Physiol. 2009 Nov 15;166(17):1950-4. doi: 10.1016/j.jplph.2009.05.001. Epub 2009 Jul 1.

 This study assessed the effect of two
precursors (l-phenylalanine and p-amino
benzoic acid) used alone or in combination
with methyl jasmonate, on the growth and
accumulation of paclitaxel, baccatin III and
10-deacetylbaccatin III in hairy root cultures
of Taxus x media var. Hicksii.
 The greatest increase in dry biomass was
observed after 4 weeks of culturing hairy
roots in medium supplemented with 1microM
of l-phenylalanine (6.2gL(-1)).
 Addition of 1microM of l-phenylalanine to
the medium also resulted in the greatest 10-
deacetylbaccatin III accumulation
(422.7microg L(-1)), which was not detected
in the untreated control culture.

 Supplementation with 100microM of l-
phenylalanine together with 100microM of
methyl jasmonate resulted in the enhancement
of paclitaxel production from 40.3microg L(-1)
(control untreated culture) to 568.2microg L(-
1), the highest paclitaxel content detected in
the study.
 The effect of p-amino benzoic acid on taxane
production was less pronounced, and the
highest yield of paclitaxel (221.8microg L(-1))
was observed when the medium was
supplemented with 100microM of the precursor
in combination with methyl jasmonate.
 Baccatin III was not detected under the
conditions used in this experiment and the
investigated taxanes were not excreted into
the medium

Curr Microbiol. 2009 Sep;59(3):227-32. doi: 10.1007/s00284-008-9270-1. Epub 2009 May 30.

 Fermentation processes using taxol-
producing fungi other than Taxus spp. may
be an alternative way to produce taxol,
which is an important antitumor agent used
widely in the clinic setting.
 In this study, a taxol-producing endophytic
fungus strain MD2 was isolated from the
inner bark of Taxus media.
 Strain MD2 produced taxol when grown in
potato dextrose liquid medium.
 The fungal taxol-which was analyzed by
ultraviolet, high-performance liquid
chromatography and mass spectrometry-was
shown to be identical to authentic taxol and
10-deacetylbaccatin III.

 Further analysis with nuclear magnetic
resonance (NMR) spectroscopy to show the
chemical structure of the fungal taxol
indicated that the fungal taxol produced an
NMR spectrum identical to that of authentic
taxol.
 Strain MD2 was identified as Cladosporium
cladosporioides according to morphology of
the fungal culture, characteristics of the
spores, and analysis of 18S rDNA sequence.
 In addition, 10-deacetylbaccatin III-10-O-
acetyl transferase gene of C. cladosporioides
MD2 was cloned for the first time and was
shown to share 99% identity with that of T. x
media and 97% identity with that of T.
wallichiana var. mairei.

galanthamine
Leucojum aestivum
Z Naturforsch C. 2009 Mar-Apr;64(3-4):219-24.

 The common effect of NH4+, NO3-, KH2PO4
and sucrose on the biosynthesis of
galanthamine by a Leucojum aestivum
shootculture was studied.
 Polynominal regression models were
elaborated for the description of the
galanthamine biosynthesis as a
consequence of variation of the
investigated variables (NH4+ between 0.20
and 0.54 g/L; NO3- between 1.44 and 3.44
g/L; KH2PO4 between 0.10 and 0.24 g/L,
and sucrose between 30.00 and 60.00 g/L).

 Optimization procedures allowed us to
establish the optimal concentrations of the
investigated variables and to propose the
modified MS nutrient medium, with 4.50
g/L KNO3, 0.89 g/L NH4NO3, 1.25 g/L
(NH4)2SO4, 0.10 g/L KH2PO4 and 60 g/L
sucrose, for the galanthamine production
by a Leucojum aestivum shoot culture.
 The proposed modified MS medium
provided considerable increase of both the
production yield and the relative content of
the target alkaloid in the alkaloid mixture.

Chem Pharm Bull (). 2008 Jan;56(1):17-21.
Solanum nigrum

 Various in vitro grown tissues (non-
regenerative callus, regenerative callus and
microshoot derived leaves) of Solanum
nigrum L. were cultured under salinity
stress (0-150 mM NaCl) for enhanced
production of solasodine, a steroidal
alkaloid and an alternative to diosgenin,
which is used as a precursor for the
commercial production of steroidal drugs.
 The role of plant growth regulators and
various concentrations of NaCl during in
vitro production of solasodine was studied.
 The in vitro yield was compared with the
yield from leaves of field grown plant.

 Solasodine content was maximum (2.39 mg/g
dry wt.) in regenerative callus when grown
on medium added with 150 mM NaCl;
followed by in vitro raised leaf of
microshoot.
 Quantitative estimation of solasodine was
carried out using a new HPTLC method,
which is validated for its recovery and
precession.
 The proposed HPTLC method showed a good
linear relationship (r(2)=0.994) in 50-2000
ng/spot concentration ranges.
 The data demonstrate that the solasodine
production in cultures was growth dependent

Hypericum perforatum
Phytochemistry. 2007 Apr;68(7):1038-45. Epub 2007 Feb 20.

 Hyperforin and adhyperforin contribute to
the antidepressant effects of Hypericum
perforatum.
 The involvement of branched-chain amino
acids in the biosynthesis of hyperforin and
adhyperforin was demonstrated in H.
perforatum shoot cultures.
 L-[U-(13)C(5)]Valine and L-[U-
(13)C(6)]isoleucine, upon administration to
the shoot cultures, were incorporated into
acyl side chain of hyperforin and
adhyperforin, respectively.

 Feeding the shoot cultures with unlabelled
L-isoleucine at a concentration of 2mM
induced a 3.7-fold increase in the
production of adhyperforin.
 The addition of 3mM L-threonine, a
precursor of isoleucine, stimulated a 2.0-
fold increase in the accumulation of
adhyperforin.
 The administration of L-valine at
concentrations of 0-5mM had no stimulating
effect on the hyperforin production in H.
perforatum shoot cultures.

 (1) Sevimli-Gur C, Cetin B, Akay S, Gulce-Iz S, Yesil-Celiktas o.
Plant Foods Hum Nutr. 2013 Sep;68(3):293-8. doi:
10.1007/s11130-013-0371-z.
 (2) Kang, Y.; Park, D.; Min, J.; Song, H.; Jeong, M.; Kim, Y.;
Kang, S.; Karigar, C.; Choi, M. In Vitro Cellular & Developmental
Biology Plant . Aug2011, 47(4), p516-524. 9p.
 (3) Barradas-Dermitz DM, Hayward-Jones PM, Mata-Rosas M,
Palmeros-Sánchez B, Platas-Barradas OB, Velásquez-Toledo RF.
Biocell. 2010;34:1–6.
 (4) Kulkarni AV, Patwardhan AA, Lele U, Malpathak NP.
Pharmacognosy Res. 2010;2:296–9.

 (5) Al-Ashaal HA. Fitoterapia. 2010;81(6):600–6.
 (6) Koslovskiĭ AG, Zhelifonova VP, Antipova TV, Zelenkova
NF. Prikl Biokhim Mikrobiol. 2010;46(5):572–6.
 (7) Chashmi NA, Sharifi M, Karimi F, Rahnama H.
Z Naturforsch C. 2010;65(5-6):373–9.
 (8) Begum F, Nageswara Rao SS, Rao K, Prameela Devi Y,
Giri A, Giri CC. Nat Prod Res. 2009;23(11):973–81.

 (9) Syklowska-Baranek K, Pietrosiuk A, Kokoszka A,
Furmanowa M. J Plant Physiol. 2009;166(17):1950–4.
 (10) Zhang P, Zhou PP, Yu LJ. Curr Microbiol. 2009
Sep;59(3):227-32.
 (11) Georgiev V, Berkov S, Georgiev M, Burrus M,
Codina C, Bastida J, Z Naturforsch C.2009;64(3-4):219–
24.
 (12) Bhat MA, Ahmad S, Aslam J, Mujib A,
Chem Pharm Bull (Tokyo) 2008;56(1):17–21.
 (13) Karppinen K, Hokkanen J, Tolonen A, Mattila
S, Hohtola A. Phytochemistry. 2007 Apr;68(7):1038-45.

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