In vitro regeneration and transformation studies on

Pelargonium graveolens (geranium) - an important

medicinal and aromatic plant

J. F. Benazir*, R. Suganthi, P. Chandrika and B. Mathithumilan

School of Biotechnology, Dr. G. R. Damodaran College of Science, Civil Aerodrome Post, Coimbatore- 641 014, Tamil

Nadu, India.

Accepted 22 September, 2013

Development of improved protocols for the regeneration of Pelargonium graveolens is important for

their commercial utilization. Callus was induced from leaf explants on Murashige and Skoog (MS)

medium supplemented with auxins and combinations of auxins and cytokinins. Micropropagation of

nodal bud explants was achieved using medium supplemented with cytokinins. The regenerated

explants were acclimatized and the transformation potential of geranium was demonstrated using

Agrobacterium tumefaciens. Maximum proliferation was obtained in medium supplemented with

combinations of 20 µM indole butyric acid (IBA) + 10 µM kinetin (KIN), 20 µM IBA + 10 µM benzyl amino

purine (BAP) and 20 µM indole-3-acetic acid (IAA) + 10 µM KIN. Plantlets were regenerated when callus

was given a photoperiod of 16 h light and 8 h dark conditions at 25°C. After being transferred to

greenhouse conditions, the plantlets were successfully acclimatized and transferred to the field

condition. The study has established an efficient protocol for in vitro regeneration and transformation of

P. graveolens as an alternative method for vegetative propagation.

Key words: Callus, geranium, in vitro regeneration, transformation.

INTRODUCTION

Pelargonium graveolens belongs to the family

Geraniaceae, and is an evergreen perennial flowering

plant commonly known for its essential oil, with rose like

aroma. The plant is commonly called Rose geranium or

Rose scented geranium and there are about 300

geranium species. It has several medicinal and aromatic

values of commercial importance (Verma et al., 2010;

Brian et al., 2010). Traditionally, geranium was used to

staunch bleeding, heal wounds, ulcers and skin disorders

as well as treat diarrhea, dysentery and colic (Matthews,

1995). The extracts of P. graveolens are reported to be

used as antibacterial and insecticidal agents (Jeon et al.,

2008; Ooshiro et al., 2009; Tabanca et al., 2013).

Geranium is now an indispensable aromatherapy oil and

is considered as balancing oil for the mind and body

(Dormon, 2000). Psychologically, geranium oil is

evidenced by its sedative, uplifting properties, which is

used to treat extreme depression, panic and anxiety.

Geranium oil is considered as one of the best remedy for

congested, oily and dry skin complications. It is also good

for minor cuts, bruises, eczemas, burns, frostbites,

chilblains, acne and broken veins. It is a vital component

in the treatment of endometriosis. Application of

geranium oil is very effective for menopausal problems,

*Corresponding author. E-mail: [email protected], [email protected]. Tel: 09442622723.

2816 J. Med. Plants Res.

diabetes, blood disorders, and throat infections and as a

nerve tonic and works well as a sedative (Bussmann et

al., 2013; Karato et al., 2006; Petrie and Peck, 2000).

The efficacy of the oil is exploited in many major food,

alcohol and beverage industries.

Rose geranium is a native of dry rocky slopes of Cape

Province in South Africa and was commercially cultivated

in France, Belgium, Spain, Morocco, Madagascar, Egypt,

Reunion Islands, Congo, China, India and the former

USSR countries (Farooqi and Sreeram, 2001). The

species was first introduced in Yercaud on the Shevroy

Hills (1370 to 1525 m attitude) of Salem district of Tamil

Nadu. Later, a number of scented and ornamental

Pelargonium species were introduced into India, some of

which got naturalized, escaped garden cultivation and are

found growing wild on Shevroy, Kodaikanal, Nilgiri Hills of

Tamil Nadu State and certain parts of Karnataka as it

prefers high altitude areas with a milder climate

(Rajeswara et al., 1999).

Commercially, geranium is widely used for scenting

soaps and high-grade perfumes due to the presence of

low molecular weight aroma compounds (Verma et al.,

2010; Farooqi and Sreeram, 2001). At one time, India

was producing about 20 tonnes of geranium oil, with an

average of nearly 1,400 hectares (Narayana et al., 1986).

However, current Indian production has decreased to

less than two tonnes per year due to significant reduction

in the availability of cultivated area and ever changing

climatic conditions. This has necessitated the application

of modern biotechnological approaches/tools to increase

the productivity to compete the requirements. The

present study aims at formulating a protocol for increased

rate of propagation and productivity of P. graveolens by

in vitro culturing and genetic transformation.

MATERIALS AND METHODS

P. graveolens saplings were obtained from Horticulture Research

Institute, Udagamandalam, The Nilgiris, Tamil Nadu. Soil from Ooty

hills fertilized with “panchkavyam” was used to grow the saplings.

The saplings were grown in shady region of the green house and

watered once a day and maintained in the department nursery.

Callus induction

The leaf explants (Figure 1A) were initially washed thoroughly with

tap water to remove the dust particles and soil residues, followed by

rinsing in soap water for few minutes. Further, the explants were

treated with 20% teepol and 10% bleach (Ala) for 10 min. Finally,

the leaves were surface sterilized by agitating them in 0.1% HgCl2

for about 5 min to eliminate fungal and bacterial contaminants

above and below the cell surface. The explants were rinsed

thoroughly in sterile distilled water. The sterile leaf explants were

cut into 5 mm long segments and plated on to Murashige and

Skoog (1962) solid medium supplemented with appropriate

combinations of nutrients and plant growth regulators (Table 1).

The bottles were sealed and incubated in a growth chamber at

25°C under 16/8 h (light/dark) photoperiod for 25 to 30 days.

Micropropagation

Micropropagation was carried out using nodal bud explants in MS

medium supplemented with auxins: cytokinins, 3% w/v sucrose and

0.7% w/v agar (Table 2). pH of the medium was adjusted to 5.8

prior to autoclaving at 121°C for 30 min. The explants were

incubated at 26 ± 1°C with 16/8 h light/dark cycles under white

fluorescent lamps (3000 to 5000 lux). Shoots were regenerated

from the callus by subjecting them in a cytokinin rich medium for

16/8 h light and dark photoperiod.

Rhizogenesis

Rhizogenesis was induced by sub culturing the in vitro regenerated

shoots into rooting medium supplemented with higher concentrations

of auxin and cytokinin and maintained under complete dark

condition.

Acclimatization

The plantlets, with shoot and roots, were taken out from the culture

medium and washed gently with double distilled water for removing

all traces of medium from the surface. The plantlets were then

transferred to small plastic cups containing sterile sand. The plastic

cups were covered with sealed plastic vinyl bags to keep full

humidity at 25 ± 2°C in light conditions (photon flux density at 25

µmol m-2 S-1, 16 h). The plantlets were moistened with water. As the

plants grew vigorous, the bags were poked with chopsticks to allow

air into the bags until the plants were self supported. The polythene

bags were removed after fifteen to twenty days. The plantlets were

later transferred to larger pots containing sterile sand and soil (1:1

ratio) and kept under shade in the green house.

Transformation studies

A 35 days old callus of P. graveolens was used for transformation

using Agrobacterium tumifaciens LBA4404 containing a binary

vector pTOK 233. Healthy compact callus from aseptically grown P.

graveolens explant was transferred into a sterile petridish

containing Whatmann filter paper. Callus was wounded by pricking

with a sterile needle all over the surface. The explants were

incubated for 48 h in MS medium to increase its meristematic

activity. Overnight grown culture of A. tumefaciens strain with pTOK

233 in yeast extract peptone (YEP) with kanamycin was directly

used for co-cultivation. Approximately 5 to 10 ml of Agrobacterium

culture were poured into a sterile petridish, 3 to 10 callus explants

were taken using sterile forceps and immersed and incubated for

10 min. After incubation, the explants were transferred to a petridish

with sterile Whatmann No.1 filter paper. Explants were washed with

MS containing 250 mg/L carbencillin to kill the Agrobacterium and

again transferred to sterile petridish with Whatmann No. 1 paper.

The washed explant was transferred to MS and incubated for 2

days in culture room. After 2 days, explants were aseptically

transferred to selection medium (MS with hygromycin and 600 µl of

250 mg/L carbenicillin). The transformed callus carrying binary

vector pTOK233 from A. tumefacie selection medium was cut into

small sections. The explant was mixed with X-gluc solution and

incubated for 2 to 12 h at 37°C in the dark. The tissue sections

Benazir et al. 2817

Table 1. Callus induction and proliferation from leaf explants of geranium.

S/No. Treatment Callus induction

(After 16 days)

Callus proliferation

(After 27 days)

Viable callus

PGR µM (After 45 days)

1 2,4-D 10-60 0 0 0

2 NAA+ 20 + + + + + Y KIN 10

3 NAA+ 20 + + N ZEA 10

4 IAA+ 20 + + + + + + + Y KIN 10

5 IAA+ 10 + + + + + Y KIN 5

6 IAA+ 20 + + + + + Y BAP 10

7 IAA+ 10 + + + + Y BAP 5

8 IAB+ 20 + + + + + + + + N KIN 10

9 IBP+ 20 + + + + + + + + Y BAP 10

0: Explants turned brown and died, +: Only induction was there, no proliferation, ++: fair growth, callus was visible to

naked eye, +++: good growth, callus covered the explants, ++++: excellent growth, prolific callus development.

were washed in ethanol. Tissues were stored in100 mM phosphate

buffer and examined under dissection microscope.

RESULTS

Callus induction and regeneration

Callus was induced from the leaf explants cultured on MS

medium supplemented with various combinations of

indole-3-acetic acid (IAA), indole butyric acid (IBA),

benzyl amino purine (BAP) and kinetin (Table 1). Curling

and appearance of protuberances occurred in a time

period of 4 to 6 days (Figure 1B). Callus induction was

observed in 16 to 17 days of incubation in dark at 25°C.

Proliferative callus was observed in 25 to 27 days of

culturing (Figure 1C). Callus induction was initiated from

the midrib and veins of the explants. Callus obtained in

dark was white friable and compact. Callus when

incubated in light turned green friable and nodular in 1 to

2 days. Pigmentation was observed in callus tissues

incubated in light after about 8 to 10 days (Figure 1C).

Maximum, proliferation was obtained in medium with

combinations of MS with 20 µM IBA + 10 µM KIN, 20 µM

IBA + 10 µM BAP and 20 µM IAA + 10 µM KIN. The

callus was maintained by sub culturing in the same

medium at an interval of 10 to 12 days. Proliferation of

sub-cultured callus was observed within a time period of

2 to 3 days (Table 1, Figure 1C and D).

Shoots were emerged as tiny pale green even globules

from 3 to 5 days old proliferative callus tissue grown

under 16/8 h light/dark photoperiod at 25°C (Table 2,

Figure 1D and E). The callus tissues were found

responsive to higher concentrations of cytokinin. The

shoot primordial developed had white petiole carrying unincised

leaf, which later showed incisions and lobes.

They were light green during the time of incubation and

turned dark green as they grew, then later on acquired

red pigmentation. Shoots without pigmentation were also

observed in callus tissues grown in complete dark

condition. Maximum proliferation and multiplication of

2818 J. Med. Plants Res.

Table 2. In vitro regeneration of shoots and roots from leaf derived calli of geranium.

PGR’s Shoot regeneration

(16 h light/8 h dark)

Rhizogenesis

(16 h light/8 h dark)

PGR µM 5 d 15 d 30 d 5 d 15 d 30d

BAP 20 + + ++ + -

← Shooting medium

KIN 20 + + + + -

BAP+ 20

+ + + + + + + + + IAA 5

KIN + 20

+ + + + + + ++ IAA 25

KIN + 10

+ + + + + + + IAA 10

KIN + 10

+ + + + + + + IBA 10

IBA + 20

← Rooting medium

- + + + + KIN 10

IBA + 20

- + +++ BAP 10

IAA+ 20

- + ++++ BAP 5

IBA + 20

- + ++++ BAP 5

++: 1-2 shootlets, +++: few shootlets, ++++: numerous shootlets, +: induction of root, visible

to naked eye, +++: proliferation, +++: maximum proliferation.

shoots was observed in hormone combinations of 20 µM

BAP + 5 µM IAA, 20 µM KIN + 5 µM IAA. After

multiplication had started, the shoots showed elongation

of petiole, which was initially white, then turned green in

color. Complete proliferation was obtained after 15 to 16

days (Figure 1E and F). Different stages of shoot buds,

from induction to whole bud formation was observed. It

appeared as small protuberance that gave rise to nodules

of globular and bent shape. These nodules on further

differentiation showed dome shaped structure with apical

incision, which were deepened further. The shoot buds

which differentiated from the callus were meristematic in

nature (Figures 1G and H).

Micropropagation

The nodal bud explants maintained in BAP and KIN

supplemented medium initiated shoot development by 3

to 4 days of inoculation. Prominent shoot multiplication

was observed around 15 to 20 days of culturing in

shooting medium. The shoot development was rapid and

covered the culture tube in 28 days. Numerous shootlets

were developed when callus tissue was sub cultured in

MS medium with 10 µM BAP + 5 µM IAA and 20 µM KIN

+ 5 µM IAA under continuous white light. All the shootlets

had green petiole with lobed leaves and glandular hairs

(Figure 1I).

Rhizogenesis

Roots were induced from the in vitro regenerated shoots

grown in medium supplemented with higher concentration

of auxin:cytokinin growth hormones (Table 2).

Small hairy fibrous roots were developed from the shoot

base after 20 to 25 days of culturing. The number of

rhizome developed was observed maximum in medium

supplemented with 20 µM IBA + 5 µM BAP (Figure 1J).

Regenerated plantlets were acclimatized under controlled

in vivo condition and transferred to pots and maintained

(Table 2, Figure 1K).

Benazir et al. 2819

Figure 1. In vitro regeneration of P. graveolens

Transformation

An effficient protocol for transfer of genomic DNA into P.

graveolens using Agrobacterium mediated transformation

was developed. After 12 h incubation at 37°C in dark,

transformed calli showed transient blue coloration while

un-transformed calli showed no sign of colorations.

Transformed calli harbored the plasmid pTOK 233 which

contain the β-glucuronidase gene, which metabolized Xgluc

and gave characteristic blue color (Figure 1L and M).

DISCUSSION

The present study aims at formulating a protocol for

increased rate of propagation and productivity of P.

graveolens by in vitro culturing and genetic transformation.

The geranium oil is an indispensable essential oil

in aromatherapy and perfume industry. Manipulation at

molecular level to develop stable transgenic plant with

increased oil production is a substitute to save the

biomass. The genetic transformation attempted as a part

2820 J. Med. Plants Res.

of this study, thus finds a significant application in

increasing the production of essential oil and fragrance

quality of this aromatic plant.

MS medium supplemented with various levels of 2,4-D

was chosen as primary medium for callus induction but

unlike the studies reported in P. graveolens (Sreedhar,

1999), the plant showed poor response and no sign of

growth. This may be due to improper modulation of

exogenous auxin and endogenous auxin content that

would prove antagonistic to callus induction. This implies

that the initiation of a proliferating culture from explants,

involves profound change in the development state of the

tissue and relates to the alterations in the basic

architecture of cells or tissues resulting in the activation

of quiescent cells (Irfan, 2001).

Curling of tissue and protuberances were formed that

was followed by the appearance of little irregular cellular

masses that were observed around the pricked end,

which may be due to exogenous substances that ooze

out of the injured tissue at cut end and stimulates cell

division (Kumar, 1992). The callus induction was

observed from the midrib as it is rich in meristamatic

tissue, and meristematic tissue have high rate of growth

as they are physiologically active (Razdan, 2003).

Substantial callus was formed in a time period of 25 to 27

days, as usually it takes 4 weeks for complete callus

formation (Kumar, 1992). The texture of callus observed

was friable and compact. Texture varies according to

explant and species used (Kumar, 1992).

Callus was colorless in dark and acquired green color

when exposed to light, which is probably due to photo

stimulation of chloroplast and development of

chloroplastids in the callus tissue. Some callus tissues

turned purple in color which may be due to accumulation

of anthocyanins (Kumar, 1992). Maximum proliferation

was obtained in medium with hormone combination MS

with 20 µM IBA + 10 µM KIN, 20 µM IBA + 10 µM BAP

and 20 µM IAA +10 µM KIN. This shows that a good

modulation of auxin and cytokinin is required for

formation of callus from geranium leaves unlike auxins

alone as reported in many cases (Razdan, 2003; Batra,

2001) (Figure 1C and D). Shoot induction occurred when

a photo period of 16/8 h light/dark was provided as

geranium is reported to be highly sensitive to light

(Sreedhar, 1999) and regeneration was delayed when

exposed to light alone (Sreedhar, 1999). The differential

coloration of shootlets was also observed. Albinos were

formed in the dark due to lack of chloroplastids while in

light, the chloroplastid differentiation was induced and

procured green color (Kumar, 1992). The pink coloration

in leaf was obtained, which can be the manifestation of

somaclonal variation (Brown and Charlwood, 1986) or

deposition of anthocyanin pigments (Razdan, 2003).

Caulogenesis occurs in blue light or low intensity white

light or in the dark with sudden exposure to light (Kumar,

1992). Shootlets grew into the medium and shows its

sensitivity to longer period of light conditions (Sreedhar,

1999). Maximum shoot proliferation was obtained in

hormone combinations MS with 20 µM BAP + 5 µM IAA

and 20 µM KIN + 5 µM IAA. This shows that the plant

requires large amounts of cytokinin with auxin

supplement for shoot proliferation (Saxena et al., 2001)

(Figure 1D to F).

Multiplication of the shoots were observed, when the

callus with shoot buds was sub cultured to MS

supplemented with 20 µM BAP + 5 µM IAA, 20 µM kin +

5 µM IAA. Saxena (2002) in her work on in vitro

procedure for micropropagation had that found MS 0.5

mg/L BAP + 0.1 mg/L NAA exhibited regeneration of

maximum number of shoots. Whereas, Sreedhar (1999)

had reported that the maximum number of shoots for in

vitro propagation of geranium was observed in medium

containing BAP and IAA at 1 mg/L.

Different stages of shoot buds from induction to whole

bud formation were observed in simple light mode of

phase contrast microscope. The stages like nodular,

dome shape bend shape, dome shapes with apical

incision were observed. The protuberances seem to

differentiate from epidermal cells and have become

meristamatic. These stages are confirmed to be stages in

shoot bud formation, as there are reports of histological

analysis of shoot differentiation in Chloroxylon swietenia.

Like in geranium, the microscopic examination of the

Chloroxylon callus from hypocotyl region revealed that

mitotic division actually initiated in pericyclic region,

giving rise to a mass of compactly arranged cells that

contained dense cytoplasm and prominent nuclei. In

pericyclic region, due to continuous proliferation of cells,

a pressure was developed from the radial growth of the

cells, which led to the disruption and degeneration of the

cortex triggering some of the newly formed cells to exhibit

re-differentiation. These cells generally occurred in nests

or nodules and could be appropriately referred to as

‘growth centres’ or ‘meristermoids’ that further

differentiated to dome shaped structure. Oval and

elongated callus cell with thick wall were also viewed in

microscopical examination. This corresponds with the

differentiation of callus tissues at the time of initiation and

further growth shows mixed population of small, more

rounded, oval and few elongated cells with dense

cytoplasm.

In geranium callus, the roots formed were small, hairy,

and fibrous and resembled that of field grown plant. The

callus tissue developed slight yellowish brown pigmentation

during rhizogenesis, (Kumar, 1992). The hormone

combination MS 20 µM IBA + 10 µM BAP resulted in the

highest amount of rhizogenesis. Similar observation was

reported by Razdan (2003), that development of roots

under light and dark suggest that the rhizogenesis in

geranium is insensitive to photoperiods and high level

of auxin favours the multiplication. Regeneration occurs

by complete organogenesis, usually initiation of shoot

buds in calli may precede rhizogenesis or sometimes it

succeeds rhizogenesis as reported by other studies

(Kumar, 1992). The latter case was observed in the geranium

callus. The callus for rhizogenesis was incubated in

the same medium (MS + 20 µM IBA + 5 µM BAP), which

gave rise to shootlets after 45 days of incubation.

Nodal buds inoculated in MS medium supplemented

with 20 µM IAA + 10 µM KIN gave rise to first, a single

shoot, then to multiple shoots, unlike the combination

given by Saxena (2001) on geranium which was 8 mg/L

KIN and 1 mg/L NAA. It took 15 to 20 days for

multiplication, unlike easy breaking of dormancy of shoot

bud; this may be due to usage of more auxin

concentration than cytokinin. The appearance of milky

white callus can also be attributed due to the auxin and

cytokinin combination with more of auxin that indeed

promotes callus induction (Razdan, 2003). The

transformed calli pieces showed transient blue coloration.

The callus was highly proliferated, and friable preincubation

requirement was only 2 days. Pre-culture is

crucial because host cell division is required for

successful Agrobacterium transformation (Binns and

Thomashow, 1988), it is not surprising that pre-culture in

a high auxin medium often enhances transformation rate

(Mathis and Hinchee, 1994; Sangwan et al., 1992). In

addition, because there was no effect of pre-culture or

transient gb-glucuronidase (GUS) expression, it is likely

that plants benefit from pre-culture by increasing the

competence of cells for DNA incorporation into

chromosome and/or de-differentiate into callus rather

than enhancing DNA transfer cells. Callus was chosen,

as it was highly meristematic compared to other explants.

As per the standardized protocol for Agrobacterium

transformation in Nicotiana tobaccum, in the present

study, the infection time and incubation time for cocultivation

is respectively 8 to 10 min and 2 days. The

same time intervals were given for geranium calli

treatment and to prevent the bacterial overgrowth,

selection on hygromycin and carbenicillin containing

medium was carried out for 2 days, which prevented the

bacterial growth. Selection was carried out in dark as

these antibiotics are light sensitive (Meilan et al., 2002).

The transient expression is a test for plasmid

transformation into calli. The plasmid pTOK 233 harbors

β-glucuronidase gene that is capable of metabolising Xgluc

and gives blue color. The transient expression even

though is a positive confirmatory test for transformation

(Jefferson, 1987) can sometimes get mislead due to

incorporation of bacteria into callus which has not been

selected properly on antibiotic medium (Han et al., 2000).

Thus we have developed a rapid protocol for the

transformation of genomic DNA into P. graveolens using

A. tumefaciens.

Benazir et al. 2821

Conclusion

The present study aimed to establishing an efficient

protocol for in vitro regeneration and Agrobacterium

mediated transformation of P. graveolens as an

alternative method for vegetative propagation. The

results suggest better methods of propagation and

efficient utilization of the in vitro protocols for the mass

multiplication of this commercially important crop.

Further, the transformation study provides a primary step

for rapid gene transfer towards the improvement of the

plant for oil content and fragrance at the molecular level.

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Verma RS, Verma RK, Yadav AK, Amit C (2010). Changes in the

essential oil composition of rose-scented geranium (Pelargonium

graveolens L Herit.ex. Ait.) due to date of transplanting under hill

conditions of Uttarakhand. Indian J. Nat. Prod. Resourc. 1(3):367-

370. 

Dear Agnieszka Ilczuk

Pls. find the attached files

https://www.ncbi.nlm.nih.gov/pubmed/10814816

http://www.crcnetbase.com/isbn/978-0-415-28487-5

http://go.galegroup.com/ps/anonymous?id=GALE%7CA129714381&sid=googleScholar&v=2.1&it=r&linkaccess=fulltext&issn=09447113&p=AONE&sw=w&authCount=1&isAnonymousEntry=true

Hoping this will be helpful

Regards

Prof. Houda Kawas

Dear

for callus initiation try to use mediums containing low ammonium and high nitrate, such as B5 medium. 

high amount of ammonium in MS medium cause toxicity due to prolonged procedure of callus formation and may hinder formed callus vitality. 

good luck

A.shomali

Dear researcher

You can see the links

http://www.bioline.org.br/pdf?tc15081

http://www.actabp.pl/pdf/2_2010/157.pdf

http://docsdrive.com/pdfs/academicjournals/ajbmb/0000/21167-21167.pdf

good luck

My thesiss about micropropagation of plargonium.l will can to help u if u have question 

Hi

I recommend to increase the BA concentration to 2 mg/L.