Novel technique for scaling up of micropropagated Ruta graveolens shoots using liquid culture...

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New Biotechnology �Volume 25, Number 1 � June 2008 RESEARCH PAPER

Novel technique for scaling up ofmicropropagated Ruta graveolens shootsusing liquid culture systems: A steptowards commercialization
Renuka Diwan and Nutan Malpathak
Department of Botany, University of Pune, Pune, Maharashtra 411007, India

Wide applications of Ruta graveolens L. in pharmaceutical industry has led to increased interest in large-

scale plant production, with emphasis on use of in vitro cultures. Earlier reports describe use of in vitro

germinated seedlings for raising shoot cultures and not regeneration. There is only a single regeneration

protocol of R. graveolens; however, it employs conventional labour intensive techniques deterring

automation. The aim of present investigation was to establish a cost effective protocol for large-scale

plant production. We report for the first time a one-step protocol with improved regeneration efficiency

for multiple shoots induction employing liquid culture systems. Effect of polyamines (putrescine and

spermine) on growth and furanocoumarin was studied. Addition of spermine enhanced the number of

multiple shoots formed (2.5-fold) and reduced the time taken by half. Spermine addition resulted in

1.47-fold in furanocoumarin production. The selected shoot line, RS2 was successfully scaled up to 5 L in

culture vessels, with 1.53-fold increase in biomass without affecting the productivity of these cultures.

This proves to be a commercially feasible alternative to bioreactors for large-scale biomass and

furanocoumarin production.

IntroductionRuta graveolens L. (Rutaceae) is a perennial medicinal plant synthe-

sizing several types of metabolites, notably flavonoids, alkaloids,

essential oils and coumarins [1–3]. Furanocoumarins (FCs) are used

in treatment of various diseases like vitiligo, psoriasis, multiple

sclerosis, cutaneous lymphomas and were recently demonstrated

to be potent anti HIV agents [4]. Because of its medicinal properties

FCs have gained wide applications in pharmaceutical industry [1].

Among various members investigated for high FC content, R.

graveolens was reported to be one of the most promising candidates

[5], as it contained high quantities of four linear, commercially

important FCs: psoralen, bergapten, xanthotoxin and isopimpi-

nellin. Therefore, interest in mass propagation of R. graveolens and

its cost reduction for commercial exploitation is gaining impor-

tance, with emphasis on use of in vitro cultures.

Earlier reports [6–8], described use of in vitro germinated seedlings

for raising shoot cultures. However, as Ruta is a cross-pollinated

Corresponding author: Malpathak, N. ([email protected])

1871-6784/$ - see front matter � 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2008.02.002

plant, shoots thus obtained would not be genetically identical to

parent plant and the genetic makeup may vary with individual

shoot. This may lead to variations in FC production. Besides, in

India the seed-set is low and seeds exhibit high dormancy. So far,

there is only a single report describing regeneration of shoots of R.

graveolens [9], which involves multiplication of pre-existing meris-

tems. De novo regeneration is preferred as there are less chances of

somaclonal variation and plants obtained are true to type. Literature

survey indicated need for optimizing the regeneration protocol.

For developing a commercially viable protocol, it is important

to maximize the number of shoots obtained and reduce the time

taken to multiply them. It has been reported earlier that poly-

amines play an important role in various physiological processes

like root, shoot formation and flower development and regulate

cell growth and embryogenesis [10]. Hence, their titers affect

explant response in vitro. Therefore, the role of exogenous poly-

amines for rapid high frequency multiples shoot induction was

envisaged and has been studied here. Polyamines are shown to be

capable of influencing secondary metabolite production as they

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RESEARCH PAPER New Biotechnology � Volume 25, Number 1 � June 2008

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act as signaling molecules during plant response to pathogen

infection (Bais et al., 2000). There are no reports on the effect of

exogenous polyamine addition on furanocoumarin production.

As polyamines regulate both growth and secondary metabolite

production we have investigated effect of their exogenous addi-

tion on Ruta shoot cultures.

All previously reported protocols involve the proliferation of

shoots via semi solid systems which making them labour inten-

sive, time consuming deterring their automation. The objective of

this work was to overcome the problems of conventional techni-

ques by exploring utility of liquid culture system that would prove

to be easier for scaling up. To avoid intensive manual handling,

automation of micropropagation by achieving scale-up of liquid

cultures has been attempted. This would prove to be commercially

feasible alternative to bioreactors.

Materials and methodsPlant materialElite plants of R. graveolens were established in the Botanic Garden,

Department of Botany, University of Pune. Preliminary experi-

ments revealed that internodal segments and leaves as the best

explants for shoot induction. Explants were washed under run-

ning water for 20 min and surface sterilized using 0.5% of Bavistin,

0.1% mercuric chloride and 70% ethanol.

Culture conditionsExplants were cultured on Murashige and Skoog’s (MS) medium

[11] supplemented with various combinations of BAP, IAA, 2,4D,

and NAA at 24 � 28C under 16 h photoperiod (40 mMol m�2 s�1).

Each treatment consisted of 15 replicates with MS basal as control.

Multiple shoots induced at the end of three weeks, were main-

tained by subculturing on liquid medium every 3 weeks. Several

shoots lines could be obtained. After seven subculture cycles the

shoot lines were screened for growth parameters. Elongated shoots

were subcultured on fresh medium to evaluate the effect of sub-

culture on shoot induction and proliferation. Biomass growth was

determined.

Biomass assayBiomass growth was assayed in terms of fresh weight, no of multi-

ple shoots formed per explant and elongation of shoots. Growth

TABLE 1

In vitro responses of selected multiple shoots lines

Combination of growth regulators used Morphogenetic

response

B1I2 (4.44 mM BAP + 11.416 mM IAA) Direct regeneration

B2I3 (4.44 mM BAP + 17.12 mM IAA) Direct regeneration

N2I3 (14.68 mM NAA + 17.12 mM IAA) Indirect regeneration

B2D2 (8.88 mM BAP + 9.04 mM 2,4 D) Indirect regeneration

MS (control) –

Results are mean of six replicates � SD.

From the numerous shoot lines obtained on various combinations of phytohormones four were

multiple shoots induced. Lines selected were RS1, RS2, RS3, RS4.

86 www.elsevier.com/locate/nbt

index (GI) and doubling time (TD) [8] were also calculated. Growth

index (GI) was calculated by using formula

GI ¼ Final weight� initial weight

Initial weight(1)

For doubling time (TD) a graph of log2 values of fresh weight were

plotted against days. Inverse of slope of any two points in expo-

nential phase gives doubling time (TD).

Polyamine additionTo study effect of polyamines on induction of multiple shoots and

growth rate, Putrescine and Spermine were added exogenously at

various concentrations on the day of initiation of experiment (day

1). Number of multiple shoots produced per explant, elongation of

shoots and growth rate were monitored for 4 weeks. To determine

the optimum concentration, polyamines were added in the cul-

ture medium to get final concentrations of 20 mM, 40 mM, 60 mM,

80 mM and 100 mM. For this 2 g of shoots were inoculated in

250 mL Erlenmeyer flasks containing 50 mL of MS liquid medium

supplemented with desired phytohormones. Polyamines were

added exogenously by filter sterilization (0.45 mM) to get the

desired final concentration. Exogenous additions of various con-

centrations for each polyamine were set up as separate experi-

ments. Flasks without polyamines were treated as control. Each

experiment consisted of three replicates and was repeated twice.

Effect on growth and furanocoumarin production was assayed

weekly for each experiment.

Furanocoumarin estimationFuranocoumarins were extracted and estimated according to a

method described by Ekiert et al. [17] with slight modifications.

In brief, plant material was shade dried and used for furanocou-

marin extraction and estimation. Hydrolyzed samples (2N HCl at

808C for 20 min) were extracted with ethanol at 808C for 20 min

followed by sonication for 20 min and then centrifuged. Filtered

supernatant was injected into a chromatographic column (RP C18,

Neucleosil), furanocoumarins were detected at 254 nm using

HPLC (Merck Hitachi) with a UV–vis detector. Solvent system

used was methanol: water (70:30) with a flow rate of 1 mL/min.

Confirmation and quantification carried out using standard Psor-

alen, Bergapten, and Xanthotoxin (Sigma-Aldrich, USA).

Frequency

of shootinduction (%)

No. of Shoots

induced/explant

Selected

line

TD days

96.3 36 � 0.17 RS1 5.7

98.8 47 � 0.54 RS2 5.2

86.4 22 � 0.09 RS3 6.5

85.3 18 � 0.86 RS4 8.9

– –

selected and screened for growth parameters like frequency of shoot induction, number of


TABLE 2

Screening of selected shoot lines for growth and FC production

Shoot lines No of multiples/

explant

Shoot

length (cm)

No of roots/

explant

Root

length (cm)

FC

(mg/10 g DW)

Productivity

(mg/g day�1)

RS1 36 � 0.23 5.0 � 0.24 2.0 � 0.53 1.2 � 0.47 113.86 � 2.4 0.47

RS2* 47 � 0.54 2.6 � 0.98 5.0 � 0.87 1.3 � 0.67 166.38 � 2.98 0.97

RS3 22 � 0.09 2.1 � 0.57 3.4 � 0.76 2.3 � 0.55 120.37 � 1.32 1.08RS4 18 � 0.86 1.7 � 0.98 1.3 � 0.87 0.8 � 0.86 47.2 � 0.92 0.33

Results are mean of six replicates � SD. *indicates selected line.

Selected lines were studied for number of shoots per explant, shoot length, number of roots per explant and root length. They were also assed for their biosynthetic potential. On the basis

of these responses, line RS2 was selected for further experiments.

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Statistical analysisEach experiment was performed with three replicates and

repeated twice with similar results. The mean � SE values have

been calculated from the data of three experiments and were

presented in the results. Statistical analysis was done for the

PLATE 1

Regeneration and scaling-up in Ruta graveolens L. Indirect and direct regeneratio

data obtained using Student’s t-test to check differences

between the treatments. The results were analysed using two-

way Anova (VassarStats) at significance level of 95% and critical

values for Turkey HSD test were calculated for significant

F-values.

n in R. graveolens L.



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Results and discussionEstablishment of shoot culturesShoot cultures obtained on medium supplemented with 2,4D, BAP,

NAA and IAA either singly or in combinations, were screened for

frequency of shoot induction, number of multiple shoots induced

perexplantand doubling time.Fromthesecultures obtainedonfour

combinations were selected. Indirect regeneration gave rise to mul-

tiple shoots on N2I3 with 22 shoots at a frequency of 86.4% (Tables 1

and 2, Plate 1a,b) followed by B2D2 giving rise to 18 shoots at a

frequency of 85.3 %. (Tables 1 and 2, Plate 1c). Indirect regeneration

has been reported in Ruta graveolens for the first time. Highest

number of shoots per explant was obtained by direct regeneration

on B2I3 with 47 shoots at a frequency of 98.8% and 36 shoots on

B1I2 at a frequency of 96.3% (Tables 1 and 2, Plate 1d,e).

Multiple shoots were excised from the explant and maintainedby

subculturing on liquid medium with same phytohormone combi-

nation. Shoots had same morphology as intact plants, without any

tendency to vitrify or develop into abnormal structures (Plate 1f,g).

Multiple shoots showed fast growth with doubling time as low as 5–

9 days. Successive subculturing had no effect on frequency of shoot

regeneration and number of shoots induced per explant.

Interestingly multiple shoots elongated readily on the same

medium. Initiation of multiple shoots, elongation and rooting

can be obtained on the same medium (Plate 1f,g). As different

media need not be supplied for each stage it cuts down cost of

TABLE 3

Effect of exogenous addition of putrescine

Treatment PUT (mM) Week FW (g) No. of m

C Wk1 1.46 � 0.45 18 � 0.92Wk2 3.45 � 0.63 23 � 0.34

Wk3 3.72 � 0.87 39 � 0.45

Wk4 4.21 � 0.68 47 � 0.98

20 Wk1 2.13 � 0.73 21 � 0.78Wk2 4.09 � 0.32 27 � 0.58

Wk3 5.15 � 0.29 42 � 0.83

Wk4 7.21 � 1.2a 51 � 1.92

40 Wk1 1.4 � 0.34 24 � 0.74

Wk2 1.47 � 0.41 32 � 0.84Wk3 2.18 � 0.53 47 � 0.31

Wk4 4.89 � 0.85 41 � 0.75

60 Wk1 2.16 � 0.74 24 � 0.69

Wk2 2.96 � 0.39 31 � 0.76Wk3 4.13 � 0.23 37 � 0.97

Wk4 5.25 � 0.98 44 � 0.92

80 Wk1 1.58 � 0.87 22 � 0.56

Wk2 1.99 � 0.21 37 � 0.83Wk3 3.42 � 0.98 42 � 0.39

Wk4 5.67 � 0.76 46 � 0.75

100 Wk1 1.62 � 0.57 18 � 0.93

Wk2 4.38 � 0.39 23 � 0.84Wk3 4.98 � 0.81 27 � 0.87

Wk4 5.32 � 0.79 29 � 0.74

WK = week, FW = fresh weight, GI = growth Index (GI = (final weight � initial weight)/initial w

Values are mean of six replicates � SD.

Exogenous addition of putrescine resulted in increase in shoot multiplication rate. Number of m

initiation could be seen at the end of first week and complete shoot elongation (2.5–3 cm) was

resulted in maximum tissue response in terms of shoot multiplication rate (Growth index (GI) =

production.

* Indicates values significant at p � 0.095 as calculated by two-way Anova (VassarStats).


production drastically and simplifies the scaling up process. Most

of the shoot cultures, reported previously [6–8], have been estab-

lished by germinating seeds in vitro and are essentially heteroge-

neous in nature with variation with individual shoot. From the

only report detailing regeneration of R. graveolens [9] it was

revealed that regeneration frequency and number of shoots per

explant (30) was comparatively lower than that obtained here and

the employed conventional techniques requiring different media

for initiation of shoots, elongation and rooting, makes it a labour

intensive and costly technique.

Selection of shoot linesFrom the shoot cultures that were established, few lines were

selected for further studies on growth parameters and their bio-

synthetic potential. Shoot lines were grown using stationary liquid

culture systems as preliminary experiments revealed that agitation

was expendable. On the basis of the growth parameters, FC pro-

duction and productivity of cultures, four lines (RS1, RS2, RS3 and

RS4) were selected which were cultured on B1I2, B2I3, B2D2, N2I3

respectively (Tables 1 and 2). Production of individual furanocou-

marin was determined and is represented in Graph 1. Bergapten

was found to be the predominant FC in almost all the shoot lines

(Graph 1). RS2 produced maximum number of multiple shoots (47

from single explant) that elongated to 2.6 cm and each shoot

produced five roots of 1.3 cm. It had the lowest doubling time

ultiples GI (%) Height (cm) FC (mg/10 g DW)

3.21 1.34 166.38 � 2.98

6.21* 2.2* 235.98 � 3.21*

a

3.89 2.43* 220.87 � 2.09a

4.25* 2.62* 213.09 � 1.98*

4.67* 2.67* 198.76 � 0.77*

4.32* 3.5* 188.32 � 0.92*

eight).

ultiple shoots produced decreased with increasing concentration of putrescine. Shoot bud

achieved at the end of third week. Exogenous addition of Put at a concentration of 20 mM

6.21, no. of multiple shoots (51) and shoot length (2.2 cm). It led to 1.41-fold increase in FC


GRAPH 1

Relative concentration of individual furanocoumarin by shoot lines. Amountof FCs produced in selected shoot lines is several fold higher than field grown

plants. In vitro shoots accumulated more bergapten as predominant

furanocoumarin.

GRAPH 2

Growth and production kinetic study of Line RS2. Kinetic studies revealed thatfuranocoumarin production took place after cultures entered stationary

phase (day 14). Production was repressed during active growth of cultures

(exponential phase).

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of 5.2 days. RS2 accumulated maximum amount of FCs

(166.38 mg/10 g DW) with a productivity of 0.97 mg/day

(Table 2). From the growth and production kinetics of RS2 it

was seen that maximum FC production took place after cultures

entered stationary phase, day 14 (Graph 2).

RS2 was used for examining effect of polyamine addition for

further increase in multiplication rate or number of multiple

shoots formed, FC production and to test its scale up potential.

TABLE 4

Effect of exogenous addition of spermine

Treatment SPR (mM) WK FW (g) No. of mu

C Wk1 1.46 � 0.65 18 � 1.3Wk2 3.45 � 0.48 23 � 1.8

Wk3 3.72 � 0.87 39 � 2.4

Wk4 4.21 � 0.92 47 � 2.5

20 Wk1 3.39 � 14 23 � 1.7Wk2 4.52 � 0.97 35 � 0.98

Wk3 4.89 � 0.93 47 � 1.7

Wk4 5.12 � 0.89 54 � 1.9

40 Wk1 2.46 � 0.93 23 � 1.7

Wk2 3.08 � 0.84 37 � 0.97Wk3 4.25 � 0.94 49 � 0.78

Wk4 5.18 � 0.89 57 � 0.76*

60 Wk1 1.42 � 0.99 25 � 0.78

Wk2 2.34 � 1.9 39 � 0.34Wk3 3.87 � 0.89 54 � 0.96

Wk4 4.94 � 0.85 65 � 0.75*

80 Wk1 2.05 � 0.87 28 � 0.83

Wk2 2.98 � 0.87 43 � 1.5Wk3 3.66 � 0.79 61 � 1.6*

Wk4 5.23 � 1.5* 72 � 2.3*

100 Wk1 2.07 � 0.84 19 � 1.0

Wk2 2.78 � 0.76 27 � 1.4Wk3 3.46 � 0.58 31 � 1.9

Wk4 4.36 � 0.70 38 � 1.7

WK = week, FW = fresh weight, GI = growth index (GI = (final weight � initial weight)/initial w

Values are mean of three replicates � SD.

Exogenous spermine addition was found to be most effective for multiple shoot initiation and

exogenous spermine added. On 80 mM of spermine, shoots initiation could be seen after 3

concentration of 80 mM was found to have a promotive effect on shoot multiplication rate (GI

increase in FC production.

* Indicates values significant at p � 0.095 as calculated by two-way Anova (VassarStats).

Polyamine additionPutrescine

Investigation of culture response to exogenous Putrescine addition

(20–100 mM) revealed 20 mM to be the optimum concentration

(Table 3). Putrescine was seen to promote growth and no of

multiples formed (Table 3). Number of multiple shoots produced

decreased with increasing concentration of putrescine. Highest

number of multiple shoots (51) were obtained on concentration of

ltiples GI (%) Height (cm) FC (mg/10 g DW)

3.21 1.34 166.38 � 2.98

4.12 1.52* 178.4 � 1.32

4.18 1.67* 203.54 � 1.94*

3.94 1.44 220.3 � 2.01*

4.23* 1.23 245.13 � 2.34*

3.36 1.5* 176.44 � 0.96

eight).

elongation. Number of multiple shoots formed increased with increasing concentration of

days and complete shoot elongation (3.5–4 cm) at the end of first week. Spermine at a

= 4.23), no. of multiple shoots (72) and shoot length (1.23 cm). Its addition led to 1.47-fold



GRAPH 3

Scaling up of RS2 (5 L). Line RS2 could be easily scaled up from 250 mL

Erlenmeyer flask to wide mouth 5 L culture vessel. Shoots formed a dense

matt and did not sink; therefore there was no need of agitation. Scaling-upresulted in 1.53-fold increase in biomass without affecting the FC

productivity.

GRAPH 4

Growth kinetics of RS2 (5 L). Growth kinetics study of RS2 in scaling up (5 L)

revealed that maximum biomass of 589 g was obtained on 90th day of

culture without loss in growth rate. Biomass obtained was 1.53-fold higherthan control. Possibility of scaling up in wide mouth culture vessel enables

large-scale shoot production and is cost effective alternative to bioreactor.

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20 mM. Here, shoot bud initiation could be seen at the end of first

week and complete shoot elongation (2.5 cm) was achieved at the

end of third week. On cultures supplemented with 100 mM of

putrescine shoots elongated to an average of 3.5 cm, however,

number of multiple shoots was drastically reduced (Table 3). More-

over it was observed that culturing multiple shoots on high con-

centrations of putrescine for prolonged durations (6 weeks) led to

loss in morphogenetic potential and led to callus formation.

Similar observations have been previously reported by Bajaj and

Rajam [12] in long-term rice callus cultures, where massive accu-

mulation of Putrescine and Spermidine resulted in near lack of

morphogenetic capacity that could be completely revived by

blocking the PA accumulation and adjusting the Put to Spd ratio.

These observations support the view that adequate PA levels and

their ratios may be important for optimum morphogenesis [12].

Putrescine at lower concentrations (20 mM) increased furano-

coumarin production to 235.98 mg/10 g DW, which was 1.41-fold

higher than control (Table 3). However, further increase in putres-

cine concentration repressed production. Bais et al. [13] reported

beneficial effects of exogenous putrescine addition in hairy roots

of Chicorium intybus where biomass production increased 2–3-fold

and coumarin production by threefold.

Spermine

Investigation of culture response to exogenous Spermine addition

(20–100 mM) revealed 80 mM to be the optimum concentration.

Number of multiple shoots formed increased with increasing

concentration of exogenous spermine added (Table 4). Maximum

number of multiple shoots 72 were obtained on 80 mM which is

1.53-fold higher than control. On addition of 100 mM of spermine

only 38 shoots were obtained as compared to 47 multiple shoots in

control. On 80 mM of spermine, shoots initiation could be seen

after 3 days and complete shoot elongation (3.5–4 cm) at the end

of first week (Table 4). This cuts down time taken for obtaining

fully elongated shoots by half as compared to control. Spermine

promoted multiple shoot initiation and elongation considerably

and no callus formation was observed. Similar promotive effects

owing to increased spermine titers on shoot multiplication rate

and shoot length in Chicorium intybus have been previously

observed [14].

Spermine also promoted FC biosynthesis (Table 4) where FC

production increased with increasing concentration of spermine.

Addition of 80 mM of spermine resulted in maximum amount of

furanocoumarins at 245.19 mg/10 g DW which was 1.47-fold

higher than control. However, 100 mM of spermine was found

to repress the FC production. Increase in coumarin production

(4.06–3.71-fold) owing to increase in endogenous levels of sper-

mine has been reported when hairy root cultures of C. intybus were

elicited by microbial agents like Pythium aphanidermatum and

Phytopthora parasitica [15].

Scaling up

Scale up in liquid culture system was attempted with the selected

shoot line RS2. Shoots were scaled up from 250 mL to 2 L in

Erelenmeyer flask. For further scale up of 5 L, large mouth glass

culture vessel was used. Shoots multiplied rapidly without any

abnormal structures or hyperhydricity. Shoots were seen float on

the surface of the medium and formed a dense matt as they grew


without sinking (Graphs 3 and 4, Plate 1g,h). Therefore, there was

no need of agitation. Highest biomass was obtained on 90th day

and was 1.53-fold higher (Graph 4). FC content was determined at

each scale up step and is represented in Graph 3. Scaling up had no

adverse effect on the biosynthetic potential of the shoot line as

seen from Graph 3, where FC production was seen to be fairly

constant during all steps. Scaling up in bioreactor requires stan-

dardization of multiple parameters which is often time consuming

and laborious. This scale up is possible without use of complicated

system and need for bioreactor. By this protocol, it was possible to

obtain multiplication of shoots on large scale with high FC pro-

ductivity. Continuous production shoots by drastically cutting

down the cost of production, gives a protocol that has definite

commercial application for mass production of shoots and can be

also explored for secondary metabolite production in vitro.

ConclusionAn improved, rapid, efficient protocol for direct and indirect

regeneration of multiple shoots for elite plants has been reported

for the first time. The protocol allows production of up to 47 shoots


aper

directly from internodal segments and up to 22 shoots indirectly

from leaves, which is much higher than the previous reports.

Induction, elongation and rooting can be obtained on the same

medium without subculture. Thus, micropropagation is one-step

protocol, which cuts down the cost of production. Selected line

RS2 was used for further enhancement in multiple shoot induc-

tion. Polyamines boosted growth and induction of multiple

shoots. Spermine was most effective and resulted in 1.53-fold

increase in number of multiple shoots formed and also reduced

the time taken by half. Exogenous polyamine addition enabled

significant enhancement in production of pharmaceutically

important furanocoumarins in in vitro shoot cultures of R. grave-

olens. RS2 could be successfully scaled up to 5 L without loss in

growth rate can be employed for micropropagation of R. graveolens

on large scale and can also be explored for commercial furano-

coumarin production in vitro.

AcknowledgementsThe Authors acknowledge financial support of University Grants

Commission (UGC), New Delhi and Dr Aditi Pant, Scientist

Emeritus, Department of Botany, University of Pune for her critical

comments during preparation of this manuscript.

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Novel technique for scaling up of micropropagated Ruta graveolens shoots using liquid culture...

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