Growth kinetics and ginsenosides production in transformed hairy roots of American ginseng--Panax quinquefolium L.

Abstract

A thin, profusely branched, fast growing hairy root line of Panax quinquefolium (American ginseng) was established by co-culturing epicotyl explants with a wild type strain of Agrobacterium rhizogenes. The transformed roots grew by over 10-fold from the initial inoculum within 8 weeks. The crude ginsenosides content in the roots was about 0.2 g/g dry wt level up to the 10th week of culture. Ginsenosides Rb2, Rd, Re, Rf and Rg1 constituted 47-49% of the crude saponin fraction between 6 and 8 weeks of growth whereas, Rc ginsenoside was accumulated only after 9th weeks when the biomass started receding. PCR amplification analysis of the hairy roots confirmed their transgenic nature by showing the presence of Ri-TL DNA with rolA, rolB and rolC genes in their genome.

Full text

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ORIGINAL RESEARCH PAPER
Growth kinetics and ginsenosides production in transformed
hairy roots of American ginseng—Panax quinquefolium L
Archana Mathur•Anita Gangwar•
Ajay K. Mathur•Priyanka Verma•
Girish C. Uniyal•Raj K. Lal
Received: 20 August 2009/Revised: 8 October 2009/Accepted: 9 October 2009/Published online: 8 November 2009
? Springer Science+Business Media B.V. 2009
Abstract
hairy root line of Panax quinquefolium (American
ginseng) was established by co-culturing epicotyl
explants with a wild type strain of Agrobacterium
rhizogenes.Thetransformedrootsgrewbyover10-fold
from the initial inoculum within 8 weeks. The crude
ginsenosides content in the roots was about 0.2 g/g dry
wt level up to the 10th week of culture. Ginsenosides
Rb2, Rd, Re, Rf and Rg1 constituted 47–49% of the
crude saponin fraction between 6 and 8 weeks of
growthwhereas,Rcginsenosidewasaccumulatedonly
after9thweekswhenthebiomassstartedreceding.PCR
amplificationanalysisofthehairyrootsconfirmedtheir
transgenic nature by showing the presence of Ri-TL
DNA with rolA, rolB and rolC genes in their genome.
A thin, profusely branched, fast growing
Keywords
Ginsenosides production ? Growth kinetics ?
Hairy roots ? Panax quinquefolium
Agrobacterium rhizogenes ?
Introduction
The roots of Panax species (Family: Araliaceae) are
valued for their triterpene saponins that have strong
immuno-modulatory, adaptogenic and anti-ageing
properties (Nocerino et al. 2000). Panax ginseng
(Koreanginseng),
P.quinquefolium
ginseng), P. notoginseng (Chinese ginseng) and
P. japonicas (Japanese ginseng) are the four major
Panax species in the trade. Nearly 30 types of
triterpene saponins, that are collectively referred to as
ginsenosides, have been identified in ginseng roots.
Among these, the ginsenosides Rb1, Rb2, Rc, Rd, Re,
Rf, Rg1 and Rg2 are considered to be the most potent
bioactives (Liang and Zhao 2008). Agri-based pro-
duction of ginseng roots is difficult and time
consuming due to extended seed dormancy, extre-
mely slow plant growth and seasonal influences (Wu
and Zhong 1999). Economic yield of roots with
required ginsenosides concentration can be obtained
only from a 4 to 7 year old crop (Johanssen 2006). To
meet the increasing market demand of ginseng roots
and pure ginsenosides, intensive efforts to produce
them in vitro in callus, cell suspensions and root
cultures are being pursued in many laboratories
(Jeong et al. 2008; Kim et al. 2009; Mathur et al.
2000, 2003; Wu and Zhong 1999). Agrobacterium
rhizogenes-mediated hairy root cultures, in particular,
are of great relevance here because of their genetic
stability and flexibility for bioreactor up scaling (Kim
et al. 2009).
(American
A. Mathur ? A. Gangwar ? A. K. Mathur (&) ?
P. Verma ? G. C. Uniyal ? R. K. Lal
Plant Tissue Culture Laboratory, Division of
Biotechnology, Central Institute of Medicinal and
Aromatic Plants, P.O. CIMAP, Lucknow 226015, India
e-mail: ak.mathur@cimap.res.in; akmcath@gmail.com
123
Biotechnol Lett (2010) 32:457–461
DOI 10.1007/s10529-009-0158-3

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We report here for the first time the establishment,
growth kinetics and ginsenoside productivity of an
A. rhizogenes-mediated hairy root line of Panax
quinquefoilum.
Materials and methods
Plant material and bacterial strains
Stratified seeds of Panax quinquefolium were pur-
chased from Mr Scott Persons, Tuckasegee Valley
(USA). They were soaked overnight in water, decoat-
ed and surface-sterilized with 1% (v/v) cetrimide for
5 min, ethanol for 1 min and then with 0.1% (w/v)
HgCl2for 3–4 min. The surface-sterilized seeds were
kept in a filter-sterilized antibiotic solution containing
500 lg chloramphenicol ml-l, 500 lg neomycin
sulfate ml-land 1,000 lg tetracycline ml-lfor 1 h
before plating for germination on an agar-gelled MS
(Murashige and Skoog 1962) basal medium. Suspen-
sions of four Agrobacterium rhizogenes strains, A4,
LBA9402, ATCC 11325 and ATCC 15834, were
raised in YMB medium (Hooykaas et al. 1977)
containing 50 mg kanamycin l-l. Root, cotyledon,
leaf and epicotyl explants (40–50 each) were excised
from the 25 to 30 day old seedlings, pricked with
needles dipped in 36 h old bacterial suspensions (OD
1.0 at 600 nm) and co-cultivated on MS medium at
28?C for 48 h. After co-cultivation the explants were
repeatedly shifted every 4th day to fresh MS medium
supplemented with 1,000 lg each of cephalexin and
ampicillin ml-lto kill LBA 9402 and A4strains or
with 300 lg carbenicillin ml-lto eliminate ATCC-
15834 and ATCC-11325 strains of the bacteria. They
were incubated in dark on an antibiotic-free MS
medium for the emergence of roots. Untreated
explants were used as control.
PCR analysis
GenomicDNAfromtransformedandnon-transformed
tissues was isolated following the method of Khanuja
et al. (1999). Plasmid DNA of A. rhizogenes was
isolated using a mini-prep plasmid kit and was used a
positive control. Oligonucleotide primers and proce-
dure described by Krolicka et al. (2001) were used for
PCRdetectionofbacterialRiplasmidgenesrolA,rolB
and rolC in the plant genome. PCR amplification with
primers homologous to the sequence 50-ATGTCGCA
AAGGCAGTAAGC-30and 50-AGTCGTACCTCGT
TGGCAGC-30(GenBank accession number X12867)
of virD gene present on Ri plasmid beyond the
transferred T-DNA segment was also done to ensure
that transformed tissue was free of A. rhizogenes cells;
Amplification conditions used were: denaturation at
94?C for 5 min, 45 cycles of 1 min denaturation at
94?C, annealing at 45?C for 1 min and extension at
72?C for 2 min followed by the final elongation for
5 min. For amplification of virD the annealing was
carriedat55?C.Theamplifiedproductswereseparated
on 1.2% (w/v) agarose gel in TEA buffer and stained
with 0.5 mg ethidium bromide l-lfor viewing under
UV light.
Growth and ginsenosides analysis
Approx. 0.2 g actively growing hairy roots (=15–
20 mg dry wt) were inoculated in 40 ml hormone-
free liquid medium consisting of one-fourth strength
of inorganic salts and double strength of vitamin
supplements of B5 (Gamborg et al. 1968) medium,
2 mg glycine l-1, and 100 mg myoinositol l-1. The
cultures were shaken at 100 rpm for 12 weeks under
diffused illumination (30 lmol m-2s-1). The fresh
and dry biomass increase was measured weekly and
growth was expressed as percent increase over the
initial inoculum weight. The freshly harvested roots
were processed for ginsenosides extraction and
HPLC analysis as previously described (Mathur et al.
1994, 2003).
Statistical analysis
Mean data obtained on nine studied parameters
namely biomass increase, crude ginsenosides content
and percent concentration of Rb1, Rb2, Rc, Rd, Re, Rf
and Rg1 ginsenosides in the crude ginsenoside pool)
was subjected to ANOVA analysis (Lal et al. 1997).
A minimum of three replicated cultures per treatment
were raised and the experiments were repeated twice.
Results and discussion
The seedling-derived root, epicotyl, leaf and cotyle-
don explants of P. quinquefolium showed differential
susceptibility for transformation when co-cultivated
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with four A. rhizogenes strains. Highest transforma-
tion frequency (46.8%) was observed when epicotyls
was infected with LBA9402 strain, followed by leaf
and cotyledon explants with LBA9402[ATC-
C158347[A4[ATCC11325. Root explants did
not respond to any bacterial strains. Co-cultivation
with bacterial strains ATCC15834 and ATCC11325
induced only limited callusing at the pricked sites
from the epicotyl tissue that did not grow further.
Such differential explants versus strain susceptibility
has also been demonstrated earlier in P. ginseng
(Chen and Punja 2002). The unorganized callus mass
originated from epicotyls infected with LBA9402
upon transfer to MS medium showed emergence of
plenty of hairy roots. For further growth and prolif-
eration the induced hairy roots were cultured in full,
half and one-fourth strength of B5 and MS liquid
media. The best proliferation was observed in one-
fourth strength of B5 medium supplemented with
double strength of vitamins, 2 mg glycine l-1, and
100 mg myoinositol l-1as was also shown for hairy
roots of P. hybrid (Washida et al. 1998). The
transformed hairy roots showed three distinct pheno-
types in culture (Fig. 1a–c): thin and highly branched
roots (TN line), thick and sparsely branched roots
(TH-line) and, thick roots with excessive callusing at
the distal ends (TH-C line). Such morphological
differences were indicative of the occurrence of
independent transformation events due to different
insertion sites of plasmid TL-DNA in the plant
genome, its copy number or T-DNA structure per se
as proposed earlier (Tepfer 1990; Mallol et al. 2001).
Out of the three different root lines, the TN line
showed best proliferation and growth due to their
healthier and better organized root tips and hence,
was advanced to detail growth and metabolite
profiling studies.
The transgenic nature of the regenerated roots was
confirmed by PCR analysis. The primers, comple-
mentary to rolA, rolB and rolC genes of TL-DNA of
the Ri plasmid generated amplification products of
approximately 770, 530 and 300 bp with the genomic
DNA of the TN hairy root line (Fig. 2a). No such
product was obtained from the non-transformed
tissue. The absence of amplification products of virD
primer (450 bp) in the transformed roots (Fig. 2b)
further confirmed that the roots were bacteria-free and
the target for the PCR amplification of rolA, rolB and
rolC genes was T-DNA incorporated into the plant
genome.
Growth kinetics and ginsenosides production in the
TN line of hairy roots was analysed over a 12 week
culture passage (Tables 1, 2). The root biomass
accumulation started from 2nd week onwards, con-
tinued up to 8 week and then steadily declined. The
cultures grew by over 4, 8 and 10 fold from the initial
inoculum after 3rd, 5th and 8th week of culture cycle.
The crude ginsenoside concentration, however, did
not vary with culture age and was steadily maintained
at around 0.2 g/g dry wt level during the growth cycle.
The initial higher content of crude saponins (0.3 g/g
dry wt) observed up to the first 2 weeks of culture
cycle might be a consequence of carry over influence
of the initial inoculum or lesser biomass gain during
Fig. 1 Hairy roots of P. quinquefolium obtained from epicotyls
explants transformed with A. rhizogenes strain LBA9402
exhibiting three different phenotypes during growth in B5
medium. a Thin branched (TN line); b Thick and sparsely
branched (TH-line); and c Thick roots with extensive callusing
(TH-Cline).Allcultureplatesshowninthefigurehadadiameter
of 90 mm
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this lag phase of growth as was suggested for hairy
root cultures of P. ginseng (Yoshikawa and Furuya
1987; Mallol et al. 2001). HPLC analysis of the crude
saponin extract of the hairy roots revealed that the
three panaxatriol type (Re[Rf[Rg1) and one
panaxadiol (Rd) type of ginsenoside constituted 47–
49% of the crude ginsenosides fraction between 6th
and 8th week of growth. Rc ginsenoside was absent
during active growth phase and appeared only after
9th week when growth declined. The Rb1 and
Rb2 ginsenosides maintained a steady level of 2–3%
of the total ginsenoside pool except a marginal
increase between 7th and 9th week of culture cycle.
The TN hairy roots line of P. quinquefolium devel-
oped in this study also maintained the ratio between
Rb and Rg ginsenosides at[1 as desired by the food
and pharma industry for field grown ginseng roots.
Interestingly, the total ginsenosides productivity of
this line was 2.3 times more than a high yielding
callus line of this species selected earlier in our
laboratory (Mathur et al. 2003; US Patent No.
6326202).
To the best of our knowledge this is the first report
of hairy root induction in American ginseng. The
transformed roots established herein would be highly
suitable for providing a sustainable production system
A
1 2
B
3
1 923456 7 810
450bp
770bp
530bp
300bp
Fig. 2 PCR analysis of thin branched (TN) hairy root line of
P. quinquefolium. a Amplified products corresponding to rolA
(lane2),rolB(lane4)androlC(lane6)genesinthetransformed
roots; lanes 3, 5, 7 showing the absence of plasmid genes in the
non-transformed tissues (negative control); lanes 8, 9, 10
showing amplification of rolA, rolB and rolC in the plasmid
DNA (positive control); b Amplification of virD gene showing
its absence (lane 1) and presence (lane 2) in the transformed
tissue and plasmid DNA, respectively
Table 1 Growth kinetics and
ginsenosides content of thin
branched (TN) hairy root line
of Panax quinquefolium as a
function of culture age
Initial inoculum used in all the
treatments was 0.2 g fresh wt
(=15–20 mg dry wt)/culture in
40 ml medium
aData are mean values of three
replications
ab Absent, tr traces
Culture
age
(week)
Dry wt increase
over the initial
inoculum (%)a
Total
ginsenosides
(g/g dry wt)
Ginsenoside fraction (% of the crude
ginsenoside pool)
Rb1 Rb2RcRdRe Rf Rg1
00 0.30.20.4ab tr4.3 0.2tr
1 450.3 0.3 0.6abAb2.9 1.3tr
2 1520.2 1.1 1.5ab 0.37.21.30.7
3 4410.21.70.5ab0.38.52.1 0.7
4 8270.12.72.7 ab 3.419.59.13.7
5 9100.22.4 2.9ab 3.920.0 9.84.6
6 9330.22.62.9ab 3.9 20.19.8 5.5
7 9950.2 2.43.3ab 4.7 23.1 10.7 5.2
81,078 0.22.83.7ab4.724.39.95.4
9 1,042 0.22.63.5 ab4.724.9 9.34.7
108300.22.61.8 3.44.928.79.4 4.6
11 7540.12.61.61.6 0.29.50.63.4
125340.1 2.20.60.8 tr9.1tr 3.1
CD at 5%12.5 0.10.20.10.01 0.10.60.3 0.1
CD at 1%16.9 0.20.30.10.010.2 0.80.40.1
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for ginseng root biomass and pure ginsenosides.
Efforts towards its bioreactor scaling up are currently
under way.
Acknowledgments
Council of Scientific and Industrial Research (CSIR), New
Delhi for carrying out this study is gratefully acknowledged.
Financialgrantreceived fromThe
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Table 2 Analysis of variance (ANOVA) for the nine parameters studied in hairy root line of Panax quinquefolium
Source of
variation
dfMean sum of sqaure
Growth
index
Crude ginsenosides
content
Rb1 Rb2Rc Rd RfReRg1
Replicates2 1620.0030.0200 0.010.04 0.060.01
Treatments12 465,609**0.28**2.4** 4.5**2.9* 13.9* 63.6**233.7**13.4**
Error24 550.01 0.01 0.0010 0.01 0.030.12 0.002
The mean values are compared with mean sum of squares between replications and treatments
* P\0.05; ** P\0.01
df Degree of freedom
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