Jordan Journal of Agricultural Sciences, Volume 3, No.

1, 2007

In vitro Propagation of Two Lavandula Species:

Lavandula angustifolia and Lavandula latifolia L. Medica
Areej A. M. Al-Bakhit, Jamal S. Sawwan* and Mohsen S. Al-Mahmoud **

ABSTRACT

A In vitro propagation of Lavandula angustifolia and Lavandula latifolia was studied. In vitro Cultures of
Lavandula angustifolia and Lavandula latifolia were established by germinating seeds of each species on
Murashique and Skoog (MS) medium supplemented with 0.5 mg/L Naphthaline Acetic Acid (NAA) incubated
for three weeks in the dark at 28 C. The best shoot multiplication rates (2.0) and highest proliferation of
Lavandula angustifolia were obtained when internodal segments were subcultured on MS medium supplemented
with 1.5 mg/L Kinetin and 0.05 mg/L NAA. Shoots were rooted on MS medium supplemented with 0.4 mg/L of
NAA or Indole Butyric Acid (IBA). Plants were successfully transferred to soil after two weeks of
acclimatization in the greenhouse. Shoot multiplication rates and proliferation of Lavandula latifolia were best
when MS medium was supplemented with 0.5 mg/L Benzyl Amino Purine (BAP), 0.05 mg/L NAA or with 1.0,
1.5 or 2.0 mg/L Kinetin and 0.05 mg/L NAA Shoots were rooted on MS supplemented with 0.3 mg/L NAA.
KEYWORDS: Lavandula, Micropropagation, (NAA), (BAP), (IBA), TDZ, Kinetin.

1. INTRODUCTION

The medicinal plant preparations play one of the key

roles in the modern pharmaceutical industry; but mass,

Lavandula is a genus of the Labiatae family.

and uncontrollable collection of medicinal plants, may

Lavandula consists of about 20 species of small

lead to the reduction of their populations.

evergreen shrubs, having aromatic foliage and flowers.

The objective of this study was to develop a protocol

Lavandula spp. are some of the most popular medicinal

for starting in vitro propagation of Lavendula spp.

herbs with great economic interest (Nobre, 1996).

The scent of most, but not all, Lavandulas is

2. MATERIALS AND METHODS

somewhat similar, namely: refreshing, herbaceous and

sweet, imparting a sense of 'clean'.

Seeds of Lavandula angustifolia were purchased from

Park Seeds; whereas, Lavandula latifolia seeds were

* Faculty of Agriculture, University of Jordan-Amman, Jordan.

** Department of Medicinal Chemistry and Pharmacognosy,
Faculty of Pharmacy, Jordan University of Science and
Technology-Irbid, Jordan. E-mail: mohsenal@just.edu.jo
Received on 8/8/2005 and Accepted for Publication on
23/1/2007.

purchased from Tisflor. (Park Seed Company 1 Parkton

Ave. Greenwood, SC 29647, USA).
Seeds of the two species of Lavandula were surface

-16 2007 DAR Publishers/University of Jordan. All Rights Reserved.

In vitro Propagation

Areej A. M. Al-Bakhit, Jamal S. Sawwan and Mohsen S. Al-Mahmoud

sterilized by washing under running tap water for 15

Media were supplemented with one of the following

minutes, and then seeds were rinsed with 95% (v/v)

synthetic

ethanol for 30 sec. The seeds were then soaked in dish

concentrations of 0.0, 0.5, 1.0, 1.5 or 2.0 mg/L and 0.05

washing detergent solution for 20 min., then soaked in a

mg/L NAA. Each growth regulator concentration was

20% commercial sodium hypochlorite (6.0% active

represented by 10 test tubes as replicates.

cytokinins;

Kinetin,

BA

or

TDZ

at

ingredient) for three times (45 min. each). Finally, seeds

Test tubes were kept in a growth room at 28C1C

were rinsed with autoclaved distilled water for three

with light regime of 16 hrs light (photosynthetic photon

times.

flux density = 40-45 mol m-2s-1), and 8 hrs dark for 10

The seeds were inoculated onto two MS media in 100

weeks. After that, the following data were recorded:

x 15mm petriplates. The first was hormone free and the

number of shoots per explant, shoot length, shoot fresh

other was supplemented with 0.5 mg/L NAA. Seeds were

weight, callus (+/-) judged by vision and callus fresh

incubated in the dark at 28 C for 14 days; the petriplates

weight. Contaminated test tubes were discarded.

were transferred from the dark to 16 hr light/8 hr dark

with 40-45 mol m-2s-1 photosynthetic photon flux

2.2 Rooting of Shoots:

The objective of the rooting experiment was to test

density and 28 C for another week for further growth.

After 21 days, germination percentage and percentage

the effect of two different synthetic auxins on rooting of

of contaminated dishes were recorded and discarded for

the two Lavandula spp. in order to complete the

each medium.

micropropagation protocol. The experiment was designed

Seedlings of the two Lavandula spp. were transferred

to fit split-split-split-plot design in which the two species

to hormone free medium for one week to minimize the

were the main classes, two synthetic auxins were tested

carry over effect of the hormone and to study the

as sub-classes; whereas, the concentrations were sub-

regeneration capacity of each species under investigation.

subclasses.
Internodal segments of 1-1.5 cm from the shoot
multiplication experiment were sub-cultured in 15 x 150

2.1 Shoot Proliferation:

of

mm test tubes, containing 15 ml of MS media

micropropagation of the two Lavandula spp., the

supplemented with one of two different synthetic auxins:

experiment was designed to fit split-split-split-plot design

NAA or IBA at concentrations of 0.0, 0.1, 0.2, 0.3, 0.4 or

in which the two species were the main classes, 2 media

0.5 mg/L. Each treatment was represented by 5 test tubes.

were tested (Murshigue and Skoog, 1962; Gamborg et

After 6 weeks the number of roots, root length and

In

order

to

test

different

protocols

shoot length were recorded.

al., 1968) as sub-classes and three synthetic cytokinins

were sub-subclasses whereas the concentrations were

2.3 Acclimatization of Plantlets:

subclasses of sub- subclasses.

Internodal segments of 1-1.5 cm from seedlings that

The plastic covers of the test tubes that contained the

were grown on hormone free media were sub-cultured in

rooted shoots from the rooting experiment were removed

test tubes containing 15 ml of either MS or B5 media.

for two days in the growth room before moving them to

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Jordan Journal of Agricultural Sciences, Volume 3, No.1, 2007

the greenhouse. In the greenhouse, rooted plantlets were

removed

from

the

medium;

roots

were

3. RESULTS AND DISCUSSION

washed

thoroughly with water to rinse away any media to avoid

3.1 Seed Germination and Plant Establishment

contamination. The plantlets were moved to pots filled

Seeds of Lavandula angustifolia, and Lavandula

with 1:1 moss peat and perlite mixture. The plantlets

latifolia were germinated successfully in vitro on MS

were irrigated immediately with a solution of inorganic

medium containing 0.05 mg/L NAA, with germination

macronutrients. Light regime of 14 hrs light and 10 hrs

percentages of 72% and 78% for Lavandula angustifolia

dark was used for 2 weeks. To maintain high relative

and Lavandula latifolia seeds respectively Table (1).

humidity, pots were placed in plastic bags; a small

Contamination percentage ranged from 7 to 13% as

opening was left on the top of the plastic bag to improve

compared to 100% for non-sterilized seeds Table (1).

air circulation. Relative humidity was reduced by gradual

Seed sterilization not only reduced the contamination

enlarging the hole. Complete removal of the plastic bag

percentage, but also enhanced the germination percentage

took place after two weeks of placement.

for the two species. Comparing the in vitro germination

percentage to that stated on the label (50%), it is noted
that in vitro seed germination percentage was much

2.4 Statistical Analysis:

The micropropagation experiment and the rooting

higher than on the label. Thus, seeds have proved to be

experiment were set to be split-split-split-plot design.

good starting material for in vitro plant establishment for

Since B5 medium did not show any growth, the media as

both L. angustifolia and L. latifolia.

sub-classes were ignored and the experiment was set as

follows: the two Lavandula spp. were the main classes,

3.2 Shoot Proliferation

the three synthetic cytokinins and a control were the sub-

Results indicate that MS was superior to B5 media

classes and the different concentrations were the sub-sub-

when supplemented with the same plant growth

classes. Data were transformed according to square root

regulators, Fig. (1). Several reports indicated that MS

for analysis (Steel and Torrie, 1980).

media was superior; Jordan et al., 1998 for Lavendula

The rooting experiment was set to be split-split-split-

dentate, Sanchez-Gras and Calvo, 1994 for Lavendula

plot design. The two Lavandula spp. were the main

latifolia and Panizza, and Tognoni, 1988 for Lavandin cv

classes, the two synthetic auxins and a control were the

Grosso. Jordan et al., (1998) suggested a protocol for

sub-classes and the different concentrations were the sub-

Lavandula dentate micropropagation by using MS

sub-classes. Data were transformed according to least

medium as the basal medium for inter-nodal segment

squared mean (Steel and Torrie, 1980).

explants. Also micropropagation of Lavandula latifolia.

Lavandula stoechas cultured on Margara medium gave

The transformed data in the experiments were

necrotic growth and the plants died later, while MS

statistically analyzed by SAS system software, and

medium did not give those symptoms (Nobre, 1996).

significance was tested according to probability test at

Contrary to others, Quazi (1980) reported the successful

5% using the Least Significant Difference (LSD).

use of B5 medium as a basal medium for multiplication

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In vitro Propagation

Areej A. M. Al-Bakhit, Jamal S. Sawwan and Mohsen S. Al-Mahmoud

(Jordan et al., 1998).

of Lavandula spp., no other authors reported similar

result in any Lavandula spp. The differences in plant

Number of shoots in both spp. was not affected by

responses and performance when cultured in different

cytokinin type and conc. Table (3). The longest shoots

culture media, may be due to the differences in osmotic

were obtained when Kinetin was used at concentrations

potential of the media, or to presence and/or absence of

of 1.0 or 1.5 mg/L (1.89 and 2.55 cm respectively) while,

specific compounds in the media (Conger, 1982).

the greatest number of internodes resulted from the use of

All tested synthetic cytokinins, along with 0.05 mg/L

Kinetin at 1.5 mg/L (4.79 internodes). For shoot weight,

NAA, showed significant effect over the control in all

all tested synthetic cytokinins gave the same shoot weight

shoot multiplication and growth parameters for the two

except for Kinetin at 0.5 and 2.0 mg/L, which gave the

Lavandula species (Table 2; Figures 2 and 3).

lowest weight Table (3).

In L.

angustifolia, the control gave significantly the least

For Lavandula latifolia, there was no significant

number of shoots, shoot length, number of internodes per

difference in number of shoots, among different

shoot and shoot weight. On the other hand, in L. latifolia,

concentrations of different tested synthetic cytokinins, but

BAP showed higher number of shoots and shoot weight

for other growth parameters the best attributes were

while in L. angustifolia TDZ gave significantly heavier

obtained at 0.5 mg/L BAP, combined with 0.05 mg/L

vegetation. Kinetin was best in the shoot length and

NAA, and at 1.0, 1.5 or 2.0 mg/L Kinetin combined with

number of internodes for both species. However, BAP,

0.05 mg/l NAA Table (3).

Kinetin and TDZ were significantly better in number of

The results of this experiment indicated that the two

shoots, shoot length, number of internodes and shoot

Lavandula species responded differently to treatments,

weight as compared to the control Table (2).

and that Lavandula angustifolia was more sensitive to

In addition, Kinetin was better in the number of

cytokinins concentrations than Lavandula latifolia. This

internodes than TDZ and the control, and was not

difference between the two species may be due to their

different from BAP. Calvo and Segura, (1988) reported

genetic make up.

that a combination of BA and IAA, or NAA gave better

Many authors reported similar differences within

organogenesis compared to IAA or NAA alone; media

species of the same genera to different treatments (Pierik,

containing 2,4-D were less effective in bud induction than

1987). Moreover, Calvo and Segura, (1989) reported that

those containing IAA or NAA for Lavandula latifolia.

explants of Lavandula latifolia, but not Lavandula

Shoots of Lavandula latifolia were proliferated from

stoechas were induced to form buds, and shoots on MS

inter-nodal

media

segments,

grown

on

MS

medium

supplemented

with

different

hormonal

combinations.

supplemented with IAA and BA, and those cultures of

IAA, or BA alone, gave no, or low shoot proliferation

Sanchez-Gras and Calvo (1996) reported that 0.05

(Sanchez-Gras and Calvo, 1996). Adult plants of

mg/L IAA and 0.8 mg BA was the best combination for

Lavandula dentata were micropropagated from nodal

shoot proliferation.

medium

Hypocotyl explants of Lavandula latifolia were

supplemented with either, BA or Kinetin with NAA

cultured on MS Medium Supplemented with 0.1 mg/L

segments

that

were

cultured

on

MS

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Jordan Journal of Agricultural Sciences, Volume 3, No.1, 2007

NAA, and 2.0 mg/L IBA for bud induction (Calvo and

number of roots were those from IBA at 0.4 mg/L (3.8

Segura,

Lavandula

roots), the longest roots were those from 0.3 mg/L NAA

angustifolia was achieved when BA exogenous levels

and 0.4 and 0.5 mg/L IBA, the longest shoots were

were 1.0 or 2.0 mg/L (Segura and Calvo, 1991); the best

obtained in 0.2 to 0.4 mg/L NAA, and finally, the largest

shoot multiplication rate of Lavandula dentata was

callus diameters were when 0.1-0.4 mg/L NAA or 0.3-0.5

obtained from 0.1 mg/L BA or 4.0 mg/L Kinetin (Jordan

mg/L IBA were used.

1989);

maximal

response

of

In vitro rooting of Lavandula stoechas shoots resulted

et al., 1998).

in 100% rooting, when basal medium contained 1.0 mg/L

NAA (Nobre, 1996). On the other hand, the number of

3.3 In vitro Rooting:

Results of the rooting experiment indicated that for

roots and root length of Lavandula vera was increased by

Lavandula angustifolia Table (4), NAA and IBA at 0.4

increasing the concentration of NAA in MS media;

mg/L gave the greatest number of roots (2.6 roots),

shoots of Lavandula latifolia and Lavandula dentata

longest roots (1.4 and 2.26 cm) and shoots (6.32 and

were rooted on MS half strength and hormone free media

6.64). For the callus diameter, 0.4 and 0.5 mg/L IBA

(Sanchez-Gras and Calvo, 1996 and Jordan et al., 1998).

gave larger diameter. As 0.5 mg/L IBA did not bring any

For Lavandula latifolia 0.3 mg/L NAA was the best.

root emergence, the large callus does not indicate a

Although this concentration of this synthetic auxin did

superior parameter. For the purpose of protocol

not give the largest number of roots but it did give better

establishment,

and

shoot length and root length as well as good callus

concentration may be weighted to be NAA or IBA at the

diameter, the reason for this choice is that plantlets of

level of 0.4 mg/L Table (4).

higher

the

best

growth

regulator

Lavandula latifolia gave different results than those

obtained in L. angustifolia. For example: the greatest

- 20 -

shoots

showed

acclimatization process.

better

survival

in

the

In vitro Propagation

Areej A. M. Al-Bakhit, Jamal S. Sawwan and Mohsen S. Al-Mahmoud

MS Media

B5 media

Figure 1: Performance of Lavandula cultures on MS and B5 media.

TDZ

Kinetin

BAP

control

Figure 2: Effect of different synthetic cytokinins (BAP, Kinetin, TDZ) over the control on shoot multiplication of
Lavandula angustifolia.

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Jordan Journal of Agricultural Sciences, Volume 3, No.1, 2007

BAP

Kinetin

TDZ

Control

Figure 3: Effect of different synthetic cytokinins (BAP, Kinetin, TDZ) over the control on shoot multiplication of
Lavandula latifolia.

Table (1): Effect of seed sterilization on seed germination and culture contamination of Lavandula angustifolia and
Lavandula latifolia.
Germination %
Spp.

Sterilized seeds

L. angustifolia
L. latifolia

72%
78%

Nonsterilized
seeds
32%
3%

Contamination %
Sterilized
seeds
13%
7%

Nonsterilized
seeds
100%
100%

Table (2): Influence of BAP, Kinetin or TDZ on number of shoots per explant, shoot length, number of internodes
and callusing (+/-) in explants of in vitro grown Lavandula angustifolia (I) and Lavandula latifolia (II) cultured on
MS medium.
Growth
regulator

No. of shoots per

explant

Shoot length (cm)

No. Of internodes
per shoot

Shoot weight (g)

Callus
(+/-)

BAP
Kinetin
TDZ
Control

I
3.05 a*
1.55 a
4.60 a
0.50 b

I
1.40 a
1.67 a
1.19 b
0.35 c

I
2.78 ab
3.05 a
2.41 b
0.60 c

I
0.48ab
0.20 b
0.90 a
0.03 c

I
+
+
+
+

II
8.5 a
4.18 b
4.25 b
0.8 c

II
1.42 b
1.71 a
1.34 b
0.53 c

II
2.66 b
3.30 a
2.59 b
1.13 c

* Means followed by different letters differed significantly (p<0.05) according to LSD-test.

- 22 -

II
1.65 a
0.16 c
0.79 b
0.029c

II
+
+
+
+

In vitro Propagation

Areej A. M. Al-Bakhit, Jamal S. Sawwan and Mohsen S. Al-Mahmoud

Table (3): Influence of different concentrations of synthetic cytokinins on number of shoots per expalnt, shoot
length, number of internodes per shoot, shoot weight and callusing (+/-) in explants of in vitro grown Lavandula
anguistifolia (I) and Lavandula latifolia (II).
Growth
regulator

No. of shoots per

explant

Shoot length
(cm)

No. of internodes per

shoot

Shoot weight (g)

Callus
(+/-)

BAP

II

II

II

II

II

0.5 mg/L (II)

1.0
1.5
2.0

1.9a*
5.0a
2.5a
2.8a

4.50a
6.10a
13.40a
10.00a

1.15cde
1.80b
1.17cde
1.15cde

2.18a
1.09c
1.30bc
1.44bc

2.80b
2.91b
2.39b
2.71b

4.09ab
2.56cd
2.61c
2.31cd

0.14ab
1.28ab
0.34ab
0.14ab

0.302b
0.860b
1.393b
4.034a

+
+
+
+

+
+
+
+

1.0a
1.9a
2.0a
1.3a

5.60a
3.80a
3.80a
3.50a

0.89de
1.89ab
2.55a
1.41bcd

1.11c
2.20a
1.98ab
2.18a

1.54b
3.09b
4.79a
2.38b

1.90d
4.11ab
4.29a
4.07ab

0.093b
0.450ab
0.173ab
0.090b

0.142b
0.127b
0.152b
0.208b

+
+
+

+
+
+
+

5.8a
3.4a
6.2a
3.0a

5.20a
3.20a
5.90a
2.70a

1.62bc
1.42bcd
1.26bcde
0.38de

1.76ab
1.63b
1.28bc
0.85d

3.35b
2.66b
2.46b
0.60c

4.42b
4.15b
2.57cd
1.48e

1.441a
1.012ab
1.028ab
0.115ab

1.064b
1.025b
0.949b
0.138b

+
+
+
+

+
+
+
+

Kinetin
0.5 mg/L
1.0
(II)
1.5 (I) & (II)
2.0
(II)
TDZ
0.5 mg/L
1.0
1.5
2.0

* Means followed by different letters differed significantly (p<0.05) according to LSD-test.

Table (4): Effect of different concentrations of synthetic auxins on rooting of in vitro grown Lavandula angustifolia
(I) and Lavandula latifolia (II) on MS medium.
Growth
regulator
NAA
0.0 mg/L
0.1
0.2
0.3 (II)
0.4 (I)
0.5

No. of roots per

shoot
I
II
0.0c*
0.0 d
0.0c
0.4 cd
0.4bc
1.0 c
0.4bc
2.2 b
2.6a
1.0 c
1.2b
0.0 d

Root length
(mm)
I
0.00 d
0.00 d
0.14 d
0.22 cd
1.40 b
0.86 bc

IBA
0.0 mg/L
0.1
0.2
0.3
0.4 (I)
0.5

0.0 c
0.0 c
0.6bc
0.4bc
2.6 a
0.0 c

0.00 d
0.00 d
0.16 d
0.38 cd
2.26 a
0.00 d

0.0 d
0.0 d
0.6 cd
1.0 c
3.8 a
1.2 c

II
0.0 d
0.10 cd
0.76 bc
1.40 ab
0.64 cd
0.00 d

Shoot length
(cm)
I
II
2.74 d
2.74 f
3.98 c
3.04 f
4.74 bc
6.92 ab
5.60 ab
7.60 a
6.32 a
6.96 ab
4.56 bc
4.98 cd

Callus diameter
(mm)
I
II
1.0 f
1.4 cd
1.2 f
2.4 a
1.8 cde
2.2 a
1.4 def
2.6 a
2.2 bc
2.0 ab
1.0 c
1.4 cd

0.00 d
0.00 d
0.44 cd
0.54 cd
1.78 a
1.66 a

2.74 d
3.96 c
4.30 c
6.40 a
4.64 bc
4.68 bc

1.0 f
1.0 f
1.8 cd
1.4 def
2.6 ab
3.0 a

2.74 f
4.46 d
4.44 de
6.02 bc
3.38 ef
4.94 cd

* Means followed by different letters differed significantly (p<0.05) according to LSD-test.

- 23 -

1.4 cd
1.2 d
1.8 bc
2.6 a
3.0 a
2.4 a

Jordan Journal of Agricultural Sciences, Volume 3, No.1, 2007

REFERENCES
Calvo, M.C. and Segura, J. 1988. In vitro morphogenesis from
explants of Lavandula latifolia and Lavandula stoechas
seedlings. Sci. Hortic. 36:131-137.
Calvo, M.C. and Segura, J. 1989. In vitro propagation of
Lavender. Hortscience 24:375-376.
Conger, B.V. 1981. Cloning agricultural plants via in vitro
techniques. CRC Press, Inc. Boca Raton, Florida, 273.
Gamborg, O.L., Millar, R.A. and Ojama, K. 1968. Nutrient
Requirements of Suspension Soybean Root Cells. Exp. Cell
Res. 50:151-158.
Jordan, A.M., Calvo, M.C. and Segura, J. 1998.
Micropropagation of adult Lavandula dentata plants. J.
Hort.Sci.Biotechn. 73 :93-96.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures. Physiol.
Plant. 15:473-497.
Nobre, J. 1996. In vitro cloning and micropropagation of

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Lavandula stoechas from field-grown plants. Plant Cell

Tiss. Org. Cult. 46:151-155.
Panizza, M. and Tognoni, F. 1988. Clonal propagation, callus
formation of plant regeneration of Lavandin. Sci. Hortic.
37:157-163.
Pierik, R. L. M. 1987. In vitro culture of higher plants. Martinus
Nijhoff Publishers, Dordecht, The Netherlands, 344.
Quazi, M. K. 1980. In vitro multimplication of Lavandula spp.
Ann. Bot., 45:361-362.
Sanchez-Gras, M.C. and Calvo, M. 1996. Micropropagation of
Lavandula latifolia through nodal bud culture of mature
plants. Pla. Cel. Tiss. Org. Cult. 45:259-261.
Segura, J. and Calvo, M.C. 1991. Lavandula spp. (Lavander):
in vitro culture, regeneration of plants and formation of
essential oils and pigments. In: YPS Bajaj (ed)
Biotechnology in Agriculture and Forestry: 15. 283-310,
Medicinal and Aromatic Plants III, Springer-Verlag, Berlin.
Steel, R. G. D. and Torrie, J. D. 1980. Principles and procedures
of statistics. 2nd ed. McGraw-Hill Book Company, Inc.

In vitro Propagation

Areej A. M. Al-Bakhit, Jamal S. Sawwan and Mohsen S. Al-Mahmoud

Lavandula angustifolia
Lavandula latifolia
*

**

Lavandula angustifolia : . Lavandula latifolia

MS 0.5/
. 28 .
Lavandula angustifolia 1.5/
0.05/ . 0.4/
. .
Lavandula latifolia 0.5/
0.05/ 1.0 1.5 2.0/ 0.05/
. 0.3/ .
: .

____________________________________________

* .
**

mohsenal@just.edu.jo :

2005/8/10 .2007/1/23

- 25 -