http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–14 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.961003 REVIEW ARTICLE Biotechnological approaches to the production of shikonins: a critical review with recent updates Sonia Malik 1,2 , Shashi Bhushan 1 , Madhu Sharma 1 , and Paramvir Singh Ahuja 1 1 Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India and 2 Department of Chemical Biology and Genetics, Centre of the Region Hana ´ for Biotechnological and Agricultural Research, Palacky ´ University, Olomouc, Czech Republic Abstract Shikonins are commercially important secondary compounds, known for array of biological activities such as antimicrobial, insecticidal, antitumor, antioxidants, etc. These compounds are usually colored and therefore have application in food, textiles and cosmetics. Shikonin and its derivatives, which are commercially most important of the naphthoquinone pigments, are distributed among members of the family Boraginaceae. These include different species of Lithospermum, Arnebia, Alkanna, Anchusa, Echium and Onosma. The growing demand for plant- based natural products has made this group of compounds one of the enthralling targets for their in vitro production. The aim of this review is to highlight the recent progress in production of shikonins by various biotechnological means. Different methods of increasing the levels of shikonins in plant cells such as selection of cell lines, optimization of culture conditions, elicitation, in situ product removal, genetic transformation and metabolic engineering are discussed. The experience of different researchers working worldwide on this aspect is also considered. Further, to meet market demand, the needs for continuous and reliable production systems, as well as future prospects, are included. Keywords Alkannin, boraginaceae, cell culture, in vitro culture, naphthoquinones, natural products, pigments, secondary metabolites History Received 21 December 2013 Revised 16 July 2014 Accepted 23 July 2014 Published online 9 October 2014 Introduction Plants are capable of synthesizing a bewildering array of chemical compounds, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, pigments, dyes, cosmetics and food additives (Lubbe & Verpoorte, 2011). These chemical compounds, commonly known as secondary metab- olites or natural products are heterogeneous compounds, generally not only required for normal growth and develop- ment of plants but increase the plant’s ability to survive and overcome local challenges by allowing them to interact with their environment (Harborne, 1993). These are produced during specific developmental stages of plant or under specific (seasonal, stress or nutritional) conditions and their production levels are often low (Malik et al., 2009, 2010a, 2013a; Verpoorte, 2000). Secondary metabolites of plant origin are in demand due to a resurgence of public interest in plant-based products. Every year, the market for plant-based products is increasing at a rate of 12–15% (Raskin et al., 2002). Table 1 summarizes the source of plant species for various secondary metabolites, their economic use and market value. Secondary metabolites are mainly classified into three main groups: polyphenols, alkaloids and terpenes. Naphthoquinones (belong to a class of polyphenols formed on a C6–C4 skeleton) have developed great interest due to their broad range of activities (Babula et al., 2009). These are widespread in nature as secondary metabolites of fungi, micro-organisms as well as plants. Naphthoquinones which include shikonin, alkannin and their derivatives, juglans and many more present a larger group of quinone pigments (Figure 1). The molecular formula of naphthoquinone is C 10 H 6 O 2 . These metabolites range from simple structures such as 5-hydroxy derivative juglone, to pigments with isoprenyl attachment such as alkannin. Shikonin, and its derivatives, are commercially the most important of the naphthoquinone pigments and this review is focused on the production of shikonin and their derivatives in plants. The main objective of this review is to highlight the reproducible methods for in vitro production of shikonins and to illustrate the different production systems for these important classes of compounds. Shikonins: their occurrence, distribution and economic value Shikonin and its derivatives are the commercially most important of the napthoquinone pigments, distributed among members of the family Boraginaceae. These include different species of Lithospermum, Arnebia, Alkanna, Anchusa, Address for correspondence: Sonia Malik, Department of Chemical Biology and Genetics, Centre of the Region Hana ´ for Biotechnological and Agricultural Research, Palacky ´ University, S ˇ lechtitelu ˚ 11, 783 71 Olomouc, Czech Republic. Tel:+420 585 632 173. Fax: +420 585 634 870. E-mail: [email protected]Critical Reviews in Biotechnology Downloaded from informahealthcare.com by UNICAMP on 10/30/14 For personal use only.
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Crit Rev Biotechnol, Early Online: 1–14! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.961003
REVIEW ARTICLE
Biotechnological approaches to the production of shikonins: a criticalreview with recent updates
Sonia Malik1,2, Shashi Bhushan1, Madhu Sharma1, and Paramvir Singh Ahuja1
1Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India and 2Department of Chemical
Biology and Genetics, Centre of the Region Hana for Biotechnological and Agricultural Research, Palacky University, Olomouc, Czech Republic
Abstract
Shikonins are commercially important secondary compounds, known for array of biologicalactivities such as antimicrobial, insecticidal, antitumor, antioxidants, etc. These compounds areusually colored and therefore have application in food, textiles and cosmetics. Shikonin and itsderivatives, which are commercially most important of the naphthoquinone pigments, aredistributed among members of the family Boraginaceae. These include different species ofLithospermum, Arnebia, Alkanna, Anchusa, Echium and Onosma. The growing demand for plant-based natural products has made this group of compounds one of the enthralling targets fortheir in vitro production. The aim of this review is to highlight the recent progress in productionof shikonins by various biotechnological means. Different methods of increasing the levels ofshikonins in plant cells such as selection of cell lines, optimization of culture conditions,elicitation, in situ product removal, genetic transformation and metabolic engineering arediscussed. The experience of different researchers working worldwide on this aspect is alsoconsidered. Further, to meet market demand, the needs for continuous and reliable productionsystems, as well as future prospects, are included.
and food additives (Lubbe & Verpoorte, 2011). These
chemical compounds, commonly known as secondary metab-
olites or natural products are heterogeneous compounds,
generally not only required for normal growth and develop-
ment of plants but increase the plant’s ability to survive and
overcome local challenges by allowing them to interact with
their environment (Harborne, 1993). These are produced
during specific developmental stages of plant or under
specific (seasonal, stress or nutritional) conditions and their
production levels are often low (Malik et al., 2009, 2010a,
2013a; Verpoorte, 2000).
Secondary metabolites of plant origin are in demand due to
a resurgence of public interest in plant-based products. Every
year, the market for plant-based products is increasing at a
rate of 12–15% (Raskin et al., 2002). Table 1 summarizes the
source of plant species for various secondary metabolites,
their economic use and market value.
Secondary metabolites are mainly classified into three
main groups: polyphenols, alkaloids and terpenes.
Naphthoquinones (belong to a class of polyphenols formed
on a C6–C4 skeleton) have developed great interest due to
their broad range of activities (Babula et al., 2009). These are
widespread in nature as secondary metabolites of fungi,
micro-organisms as well as plants. Naphthoquinones which
include shikonin, alkannin and their derivatives, juglans and
many more present a larger group of quinone pigments
(Figure 1). The molecular formula of naphthoquinone is
C10H6O2. These metabolites range from simple structures
such as 5-hydroxy derivative juglone, to pigments with
isoprenyl attachment such as alkannin. Shikonin, and its
derivatives, are commercially the most important of the
naphthoquinone pigments and this review is focused on
the production of shikonin and their derivatives in plants. The
main objective of this review is to highlight the reproducible
methods for in vitro production of shikonins and to illustrate
the different production systems for these important classes
of compounds.
Shikonins: their occurrence, distribution andeconomic value
Shikonin and its derivatives are the commercially most
important of the napthoquinone pigments, distributed among
members of the family Boraginaceae. These include different
species of Lithospermum, Arnebia, Alkanna, Anchusa,
Address for correspondence: Sonia Malik, Department of ChemicalBiology and Genetics, Centre of the Region Hana for Biotechnologicaland Agricultural Research, Palacky University, Slechtitelu 11, 783 71Olomouc, Czech Republic. Tel:+420 585 632 173. Fax: +420 585 634870. E-mail: [email protected]
Vanilla planifolia Impeller driven reactor 72 ESCAgenetics USA
Large-scale commercial production of secondary compounds from in vitro culture is recognized for shikonin, ginsenosides, berberine and taxol. c: cellculture; r: root culture; h: hairy root culture.
Data collected from Malik et al. (2011a) and Nosov (2012).
4 S. Malik et al. Crit Rev Biotechnol, Early Online: 1–14
feasible as it involves twelve step reactions and the final yield
is only 0.7% (Terada et al., 1983). In order to find an
alternative plant source to meet the still emerging demand
of this product, other members of the family Boraginaceae
were also exploited for their potential to produce shikonin
derivatives.
Pietrosiuk et al. (1999) isolated shikonin derivatives and
pyrrolizidine alkaloids from callus and cell suspension
cultures of A. euchroma. The presence of acetylshikonin
and b-acetoxyisovalerylshikonin in cell suspension cultures of
A. euchroma has been reported by our group (Sharma et al.,
2008). The cell culture of A. hispidissima was found to
produce arnebins (Jain et al., 1999). There are reports on the
production of shikonin derivatives in callus cultures of
Echium lycopsis (Fukui et al., 1983; Inouye et al., 1981).
Isolation procedures for isohexenylnaphthazarin compounds
from callus and cell suspension cultures of A. euchroma have
been demonstrated by Damianakos et al. (2012).
Methods for improving productivity
Although plant cells are capable of producing secondary
metabolites under in vitro conditions, until now only a few
plant metabolites have been commercialized or reached at
industrial scale through cell culture. In order to improve the
yield for commercial exploitation, efforts were focused on
Figure 3. Regeneration in A. euchroma. (a) Shoot cultures with vitrified leaves (arrow marked) on 20.0 lM TDZ supplemented medium. (b) Shoot budregeneration (arrow marked) from intact leaves (attached to shoots) on 5.0 lM TDZ. (c) Growth of shoot buds on 5.0 lM TDZ. (d) A part of swollenleaf with protuberances (arrow marked) and scattered vascular bundles. (e) Initiation of shoot bud (continuation of vascular strand with the motherexplant). (f) Shoot bud showing shoot apex (SA), subtending leaves (SL) and vascular strand (VS) in continuity with the mother explant (arrowmarked). (g) Rooted plantlet on 0.25mM IBA after 20 days. (h) Hardened plants under greenhouse conditions. Reprinted from Malik et al. (2010b)Copyright (2010) International Federation for Cell Biology, with permission from John Wiley and Sons.
Figure 4. A. euchroma callus (a) induction and (b) proliferation on MSmedium with BAP (10.0 mM) + IBA (5.0mM).
DOI: 10.3109/07388551.2014.961003 Biotechnological approaches to the production of shikonins 5
production. However, cell growth seems to be hampered at
extremes of pH (Malik et al., 2008; Figure 8). The higher
yield of these pigments at alkaline pH was correlated to
enhanced activity of geranyltransferase, a key regulatory
enzyme, which showed optimum activity at pH 7.1–9.3
(Heide & Tabata, 1987a,b).
Optimization of culture conditions
The biosynthesis and accumulation of secondary compounds
is influenced by the photoperiod, quality and intensity of light
and temperature. Under in vivo conditions, shikonins are
accumulated in underground parts of the plants. The in vitro
cultures of L. erythrorhizon also showed similar behavior, as
pigment formation was inhibited under light conditions
(Yazaki et al., 1997a, 1999).
Heide et al. (1989) observed the reversible effects of light
on shikonin biosynthesis and suggested that cell cultures of
L. erythrorhizon can act as a model to study the light
regulation of shikonin biosynthesis. Tabata et al. (1993)
suggested that light inactivates the flavoprotein necessary for
an enzymatic process leading to shikonin by decomposing the
cofactor flavin mononucleotide (FMN) into lumiflavin. Later,
Yazaki et al. (2001) characterized the dark inducible genes
from L. erythrorhizon (LEDI) and found the regulatory role
of LEDI-II for shikonin biosynthesis. The inhibitory effects
of light on shikonin biosynthesis were also elucidated in
E. lycopsis (Fukui et al., 1983), O. paniculatum (Liu et al.,
2006), O. echiodies (Lattoo, 2005) and A. euchroma (Malik
et al., 2011b; Figure 9). Optimum production of shikonin
derivatives from cell culture of L. erythrorhizon was observed
at 20–25 �C (Tabata & Fujita, 1985). Also, Malik et al.
(2011b) reported maximum pigment production in
A. euchroma at 25 �C (Figure 10).
Two-stage culture medium
Fujita et al. (1981a) found enhanced production of shikonin
derivatives in M-9 medium from L. erythrorhizon cell culture
with poor biomass yield. Therefore, a two-stage culture
process was employed using modified LS medium (MG-5) for
cell growth and modified WH (M-9) for shikonin derivatives
production (Tabata & Fujita, 1985). Also, in cell suspension
cultures of A. euchroma, a two stage culture media approach
was adopted due to non-linked behavior of cell growth and
0
20
40
60
80
100
120
140
160
180
5 5.75 6.5 7.25 8.758 9.5
pH
Cel
l Bio
mas
s (g
, Fw
.L−1
)
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
0.450
0.500
Pig
men
t Con
tent
(O
D62
0)
Cell Biomass
Pigment
Figure 8. Effect of pH on shikonin derivatives production and average cell biomass yield during cultivation of A. euchroma cell suspension (±SD).Reprinted by permission from Malik et al. (2011b). Copyright (2011) International Federation for Cell Biology.
DOI: 10.3109/07388551.2014.961003 Biotechnological approaches to the production of shikonins 7
shikonin derivatives production. Cells were first cultured
for 8 days in a liquid MS + BAP (10.0mM) + IBA (5.0 mM)
medium for cell growth (Growth Medium) and then
transferred to shikonin derivatives or pigment production
medium (APM; Malik et al., 2008).
Addition of precursors and adsorbents
The addition of a precursor, phenylalanine has been reported
to increase the accumulation of shikonins in a cell suspension
culture of L. erythrorhizon (Mizukami et al., 1977). Fukui
et al. (1984) observed that cells of L. erythrorhizon, cultured
in M-9 medium containing activated charcoal, started
producing an orange color benzoquinone derivative, echino-
furan B instead of shikonin. The presence of an adsorbent
PVP (1.0 g/l) in cell suspension culture of A. euchroma was
found to promote cell growth and shikonin derivative
production (Zakhlenjuk et al., 1992).
Use of elicitors/inducers
Elicitation is one of the strategies employed in plant cell
culture to improve the productivity of secondary metabolites
(Namdeo, 2007). Endogenous polysaccharides were found to
induce the biosynthesis of shikonin derivatives in cell cultures
of L. erythrorhizon (Fukui et al., 1990). Ge et al. (2006)
reported the use of rare earth elements in cell cultures of
A. euchroma for enhanced cell growth and shikonin derivative
production. In order to enhance the shikonin content in cell
culture of A. euchroma, fungal elicitors were reported by
Fu & Lu (1999). They obtained 2.24 times higher production
of shikonin derivatives from cells on addition of Rhizopus
oryzae in the medium. Ning et al. (1994) observed the higher
production of shikonin derivatives in cell suspension cultures
of Onosma paniculatum on addition of fungal elicitor. There
are reports of enhanced production of shikonin derivatives in
cell culture of boraginaceous plants by methyl jasmonate.
Enhanced production of shikonin was observed from cells of
L. erythrorhizon (Gaisser & Heide, 1996) and A. euchroma
(Bychkova et al., 1993) and there was an increase in alkannin
pigment, an enantiomer of shikonin from Alkanna tinctoria
cells (Urbanek et al., 1996) in response to methyl jasmonate.
Urmantseva et al. (1999) reported that methyl jasmonate
induced shikonin biosynthesis in cells of A. euchroma by
activation of phenylalanine ammonia-lyase. The elicitation
activity of methyl jasmonate was higher than that of yeast
extract (Mizukami et al., 1992, 1993). Touno et al. (2005)
studied the role of ethylene on shikonin biosynthesis in stem
cultures of L. erythrorhizon.
Permeabilization
Permeabilization of the cell membrane enables the passage of
various enzymes or molecules into and out of the cell. Chung
et al. (2006) studied the effect of gamma-irradiation on
shikonin derivatives production from callus cultures of
L. erythrorhizon. A brief exposure of cells to ultrasound
resulted in a 60–70% increase in the yield of shikonin
derivatives in L. erythrorhizon (Lin & Wu, 2002). It has been
reported that the addition of 1–2% (w/v) Na2EDTA and 1–5%
(v/v) Triton X-100 at the end of growth phase stimulated
the accumulation of shikonin derivatives in cell suspension
cultures of A. euchroma (Zakhlenjuk et al., 1992).
Immobilization
Immobilization can be an effective tool to improve the
effectiveness of a plant cell production process (Dornenburg,
2004). Kim & Chang (1990b) showed 2.5 times higher
production of shikonin from cells of L. erythrorhizon by
immobilization using calcium alginate.
In situ product removal and two-phase culture
Use of a two-phase culture system to improve secondary
metabolites production from different plant species, its types
as well as applications have been described in a recent review
by Malik et al. (2013b). In situ extraction of shikonin in cell
suspension culture of L. erythrorhizon using n-hexadecane as
lipophilic phase was reported by Deno et al. (1987).
Shimomura et al. (1991) and Sim & Chang (1993) success-
fully employed this method in hairy roots of L. erythrorhizon
(Bruce & Daugulis, 1991). The time period for addition
of organic solvent affects the cell growth and secondary
metabolite production. The addition of n-hexadecane to the
0 100 200 300 400 500 600 700
2
4
6
8
10
12
14
Day
s
Shikonin derivatives (µg/g FW)
20 °C 25 °C 30 °C
c
a
b
a
a
a
a
b
b
b
b
bb
cc
cc
cc
Figure 10. Effect of temperature on shikonin derivatives production incell suspension cultures of A. euchroma (± SD). Different letters column(a, b, c) on the bars showing the significant difference (Tukey test,p� 0.05). Reprinted from Malik et al. (2011b) Copyright (2011)International Federation for Cell Biology, with permission from JohnWiley and Sons.
Figure 9. Cell suspension cultures of A. euchroma under (a) light and(b) dark conditions after 8 days of culture. Reprinted from Malik et al.(2011b) Copyright (2011) International Federation for Cell Biology, withpermission from John Wiley and Sons.
8 S. Malik et al. Crit Rev Biotechnol, Early Online: 1–14
Table 3. Chronological studies on in vitro culture of shikonin bearing plant species.
Year References
1974 Shikonin derivatives obtained from callus cultures of Lithospermum erythrorhizon. Tabata et al. (1974)1978 Stable cell lines in L. erythrorhizon with improved growth and pigment production obtained using cell-
aggregate cloning technique .Mizukami et al. (1978)
1981 Effect of different basal media was studied on cell growth and shikonin derivatives production in cellsuspension culture of L. erythrorhizon.
Fujita et al. (1981b)
1981 A new medium developed for the production of shikonin derivatives from cell suspension culture ofL. erythrorhizon.
Fujita et al. (1981a)
1981 Production of shikonin derivatives in callus cultures of Echium lycopsis. Inouye et al. (1981);Fukui et al. (1983)
1983 Analysis of shikonin derivatives in cell suspension cultures of L. erythrorhizon. Fujita et al. (1983)1983 Commercial production of shikonin derivatives achieved from cell culture of L. erythrorhizon. Fujita (1988a)1987 Tissue culture of Arnebia euchroma initiated.1987 Factors affecting the production of shikonin derivatives in callus and cell suspension culture studied
in L. erythrorhizon.Hara et al. (1987)
1989 Reversible effect of light on shikonin biosynthesis in L. erythrorhizon. Heide et al. (1989)1991 Two phase culture system for the extraction of shikonin derivatives from hairy roots of L. erythrorhizon. Shimomura et al. (1991);
Sim & Chang (1993)1993 PFP-resistant cell lines selected in A. euchroma. Zakhlenjuk et al. (1993)1996 Cell lines of A. euchroma selected during different stages of culture by in vitro screening method. Sokha et al. (1996)1996 Important enzymes involved in regulation of shikonin biosynthesis were identified. Gaisser & Heide (1996);
Lange et al. (1998)1997 Effects of elicitor methyl jasmonate were investigated on shikonin biosynthesis in cell culture of
L. erythrorhizon.Yazaki et al. (1997b)
1997 Effect of various auxins and cytokinins on organogenesis and embryogenesis in selected line ofL. erythrorhizon.
Yu et al. (1997)
1999 Synergistic effect of in situ extraction and elicitation on shikonin derivatives production in cellsuspension cultures of A. euchroma.
Fu & Lu (1999)
1999 Isolation of naphthoquinones and pyrrolizidine alkaloids from callus and cell suspension culturesof A. euchroma.
Pietrosiuk et al. (1999)
1999 Arnebins showing antibacterial and antifungal activity were found in cell culture of A. hispidissima. Jain et al. (1999)1999 Studies were carried out to modify the biosynthetic pathway for enhanced production of shikonin
derivatives.Sommer et al. (1999)
2000 Cell suspension culture of L. erythrorhizon were found to produce different groups of compounds. Yamamoto et al. (2000)2001 Regulatory mechanism of light was studied on formation of shikonin derivatives in L. erythrorhizon. Yazaki et al. (2001)2001 Direct regeneration from leaves of A. euchroma. Ji & Wang (2001)2001 High shikonin content in the selected p-fluorophenylalanine resistant callus lines in A. euchroma. Bulgakov et al. (2001)2002 Production of alkannin from hairy roots of A. hispidissima. Singh et al. (2002)2002 Enhancement in production of shikonin derivatives in cell suspension culture of L. erythrorhizon by using
low energy ultrasound.Lin & Wu (2002)
2002 Different shikonin derivatives were observed in cell suspension cultures of L. erythrorhizon. Yamamoto et al. (2002)2005 Direct regeneration from cotyledons and hypocotyls in A. euchroma. Jiang et al. (2005)2005 Shoot regeneration and somatic embryogenesis from leaf derived callus in A. euchroma. Manjkhola et al. (2005)2008 Role of pH on shikonin derivatives production in cell suspension culture. Malik et al. (2008)2008 Presence of acetylshikonin and b-acetoxyisovaleryl shikonin in cell suspension cultures of A. euchroma. Sharma et al. (2008)2009 Shikonin formation in cell suspension culture of Onosma paniculatum was reported to be regulated by
Nitric oxide.Wu et al. (2009)
2010 In vitro induction of hairy root culture and shikonin production in A. hispidissima. Chaudhury & Pal (2010)2010 Direct plant regeneration, micropropagation and shikonin induction in A. hispidissima. Pal & Chaudhury (2010)2010 The importance of pre-culture time and concentration of TDZ for direct regeneration from in vitro leaves
of A. euchroma.Malik et al. (2010b)
2010 Pathway for shikonins biosynthesis was identified. Singh et al. (2010)2010 A two-phase liquid culture system using liquid paraffin was established to elicit shikonin and alkannin
derivatives in E. italicum.Zare et al. (2010)
2011 Different physico-chemical factors affecting the shikonin derivatives production in cell suspensioncultures of A. euchroma were studied.
Malik et al. (2011b)
2011 Micropropagation and establishment of callus and cell suspension culture of A. hispidissima foroptimization of alkannin production.
Shekhawat & Shekhawat(2011)
2011 Shikonin production potential of E. italicum callus. Zare et al. (2011)2011 LeERF-1, a novel AP2/ERF family gene within the B3 subcluster was down-regulated by different
light conditions in L. erythrorhizon.Zhang et al. (2011)
2012 Enhanced production of shikonin derivatives in hairy roots of L. canescens. Sykłowska-Baranek et al.(2012)
2013 In vitro propagation and genetic fidelity of micropropagated plants was assessed for A. hispidissima. Phulwaria et al. (2013)2013 The effects of fungal elicitor and macroporous adsorption resin were studied on shikonin accumulation in
hairy roots of A. euchroma.Zhang et al. (2013)
2013 Shikonin production from cell suspension culture of Arnebia sp. and it’s up-scaling through bioreactor. Gupta et al. (2013)
10 S. Malik et al. Crit Rev Biotechnol, Early Online: 1–14
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Genetic transformation and transformed plants
A low yield of secondary compounds from plant tissues under
in vitro conditions is a major bottleneck in their production at
the commercial level (Charlwood & Pletsch, 2002). Through
genetic engineering, it is possible to manipulate the regulatory
steps of the biosynthetic pathway in order to increase the yield
of required compounds (Verpoorte et al., 1999). There are
reports on the transformation of shikonins bearing plants with
Agrobacterium rhizogenes to enhance the secondary metab-
olite content (Fukui et al., 1998; Shimomura et al., 1991;
Yazaki et al., 1998). Yazaki et al. (1998) obtained transfor-
mants in L. erythrorhizon using A. rhizogenes strain 15 834
with stable and higher production of shikonin derivatives.
Shimomura et al. (1991) transformed the shoot cultures of L.
erythrorhizon with A. rhizogenes strain 15 834 and found a red
pigment after 2–3 weeks culturing on agar gelled and liquid
root culture media. Similarly in L. canescens, hairy roots were
shown to produce ca. a 10% higher content of acetylshikonin
and isobutrylshikonin as compared to natural roots (Pietrosiuk
et al., 2006). Hairy root transformation for other genera of
the Boraginaceae family has also been reported. In another
report by Singh et al. (2002), alkannin was obtained on a
half-strength agar solidified MS medium from hairy roots
of A. hispidissima induced with A. rhizogenes strain 15 834.
Sommer et al. (1999) modified the biosynthetic pathway
to 4HB in hairy root cultures of L. erythrorhizon by
introducing bacterial gene ubiC but it did not show any
significant increase in yield of shikonin derivatives in
transformed plants. Boehm et al. (2000) made an attempt to
manipulate the biosynthetic pathway of secondary metabol-
ism by the introduction of the bacterial gene ubiA in hairy
root cultures of L. erythrorhizon. The transformed hairy roots
showed high ubiA activities, however, ubiA overexpression
was not found sufficient to increase the shikonin content. In A.
euchroma, callus cultures were transformed with
pCAMBIA1302 containing HMGR cDNA (unpublished
data). The cell suspension cultures, raised from transformed
callus, did not show significant alteration in the shikonin
derivative content as compared to the control. Despite high
activity of HMGR, the shikonin content remained unchanged.
A high activity of HMGR in L. erythrorhizon hairy root
cultures was reported by transformation with A. rhizogenes
containing HMGR and ubiC genes under the control of
All the authors have read the manuscript and there are no
conflicts of interest.
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