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EX-SITU CONSERVATION OF LAVANDULA ANGUSTIFOLIA

USING IN VITRO TECHINQUES

Leelavathi D

MES COLLEGE,

MALLESWARAM,

BANGALORE -03



EX-SITU CONSERVATION OF LAVANDULA ANGUSTIFOLIA

USING IN VITRO TECHINQUES

Leelavathi D

M.E.S COLLEGE, MALLESWARAM, BANGALORE-560003

Lavandula angustifolia L. is a perennial shrub belonging to the family Lamiaceae. It is in great demand for the

lavender oil it yields which is used in perfumery, cosmetics, flavouring and pharmaceutical industries. In order to

meet the growing demand of lavender oil, in vitro techniques are being used as alternative method for large scale

multiplication and ex-situ conservation. In the present investigation, in vitro axillary bud explants of Lavandula

angustifolia were cultured on MS basal medium supplemented with BAP 8.88µM and NAA 2.68µM to induce

multiple shoots. Further, these shoots were subcultured on the same medium to produce more number of multiple

shoots. Well developed multiple shoots were cultured on MS basal medium fortified with BAP 8.88µM, IBA

4.92µM and NAA 2.68µM for root formation. The hardened regenerated plant were acclimatized plants were

transferred to soil with 90% survival frequency.

The in vitro axillary buds were used for synthetic seed production using Sodium alginate and Calcium

chloride as matrix and complexing agent for encapsulation of axillary buds of Lavandula angustifolia.

Key Words: Lavandula angustifolia L., In vitro propagation, hardening and encapsulation.

Abbreviations: MSBM - Murashige and Skoog’s Basal Medium

BAP - 6- Benzyl Amino Purine

2, 4-D - 2, 4- Dichlorophenoxy acetic acid

NAA - Napthalene acetic acid

IBA - Indole Butyric acid

Kn - Kinetin



AIM:

The objective of this experiment was to explore the possibility of using aseptic vegetative propagules for large scale

multiplication and synthetic seed production, which can be used as a tool for ex- situ germplasm conservation of

aromatic plant like L.angustifolia.

INTRODUCTION

Lavandula species are of great interest due to their content of essential oils, which are important to the perfume,

cosmetic, flavoring and pharmaceutical industries. They are also grown as ornamental plants. Species of lavender

leaves have spasmolytic action on the guinea pig ileum. Lavender is also used as carminative, spasmolytic, diuretic,

antidepressant, rheumatism, toothache, sinusitis, stress and migraine. Both fresh and dried lavender flowers are used

in herbal pillows, lavender bags, household cleaning products and scented candles (Lis-Balchin and Hart 1999).

It has antimicrobial activity against Enterococcus faecalis and also possesses antifungal properties, e.g. against

Aspergillus niger and Fusarium culmorum (Lis-Balchin et al., 1998). Lavender and lavandin oil have long been the

major essential oils produced on the southern slopes of the French Alps. The world production of high quality

lavender oil is about 200 tonnes/ year. The world production of lavandin oil is about 1000 tonnes /year. Bulgaria

produces 100-129 tonnes, France 55, USSR35 tonnes and US exports 121 tonnes.

Lavandula angustifolia L. (Lavender) belongs to the class Dicotyledon order Tubiflorae, Family Lamiaceae it

comprises a quadrangular stem with young branches, opposite and entire leaves, greyish-green in colour. The leaves

are almost linear to oblong-lancoelate, usually tomentose. The upper branches bear terminal spikes. The part of the

plant above the ground is covered with hairs and glands that emit an aromatic fragrance (Duke, 1985).

The genus consists of 32 species. They are distributed from the Canary Islands, Maderia, Mediterranean Basin, North

Africa, South West Asia, Arabian Peninsula and tropical NE Africa and India (Lis-Balchin, 2002a).

MATERIAL AND METHODS

In vitro seedlings of Lavandula angustifolia were raised through seed germination on the nutrient medium. These

were used as mother plants for further tissue culture studies. The seeds were immersed and washed in a solution of

Labolene (1:20) for 10 minutes, rinsed in sterile water and transferred to 70% alcohol for 30 seconds, followed by



0.1% of mercuric chloride (HgCl2) for one minute. They were then rinsed in sterile water to remove the traces of

sterilant and inoculated on MS basal medium fortified with BAP (8.88μΜ) under aseptic conditions. pH of the

medium was adjusted to 5.6 before autoclaving at 1210c for 15 minutes and the cultures were kept at 260c± 20c under

white fluorescent light at a photon density of under photoperiodic regime of 16 hours light and 8 hours dark cycles.

OBSERVATIONS

Axillary bud explants measuring 0.5cm with stem were excised from 20 days old in vitro plants of Lavandula

angustifolia and cultured (Fig. 1) on MSBM fortified with BAP with different concentrations ranging from 4.44μΜ,

6.66 μΜ, 8.88 μΜ, 11.11 μΜ, 13.32 μΜ and Kn ranging from 4.64 μΜ, 6.96 μΜ, 9.28 μΜ, 11.60 μΜ, 13.92 μΜ and

NAA 2.68 μΜ separately to study their effect on axillary bud multiplication (Table1).

After 10 days of culture, shoot initiation with 2-3 leaves (Fig. 2) were observed on all the concentrations of growth

regulators studied with varying percentage (46-91%) of response (Table 1,Graph 1.). The highest (91%) and lowest

(46%) percentage of response was observed on MSBM +BAP (8.88 μΜ) + NAA (2.68 μΜ) and MSBM +Kn (13.92

μΜ) + 2, 4-D (2.26 μΜ) respectively.

After 20 days of culture 2-3 multiple shoots were noticed (Fig.3). After 27 days of culture 3-5 elongated shoots

(Fig.4) were observed. After 36 days of

culture, the shoots were subcultured on the same medium to obtain more number of multiple shoots.

After 14 days of subculture, 6-8 multiple shoots were observed (Fig.5) on MSBM +BAP (8.88 μΜ) + NAA (2.68

μΜ) 10-12 multiple shoots (Fig.6) were formed after 28 days of subculture. 32-36 elongated multiple shoots which

attained the height of 3-5 cm (Fig.7) were noticed after 42 days of subculture. These shoots developed roots when

inoculated on rooting medium on MSBM + NAA (2.68 μΜ) + IBA (4.92 μΜ) after 28 days of culture (Fig.8). The

axenic plants were subjected to hardening (Fig.9).

In the present investigation, the statistical analysis of the data revealed highly significant differences existing

between and within the treatments. The mean number of shoots per explant ranged from 18.30-36.50(Table1,

Graph 1). The highest mean number 36.50 was observed on MSBM +BAP (8.88 μΜ) + NAA (2.68 μΜ) and

the lowest mean number 18.40 on MSBM +Kn (13.92 μΜ) +2, 4-D (2.26 μΜ) respectively.

In case of Lavandula angustifolia in vivo plant materials like leaf and stem and in vitro plant material like stem and

leaves was subjected for GC analysis, here 1gm of the dried plant material was dissolved in methanol and was

analysed for its chemical composition and the constituents were identified by comparing them with those in the

standard. GC analysis of the plant material revealed the presence of linalool compound as major constituent with

0.004 % with a retention time of 11.961 with an area of 129359 as shown in the figure in vitro and with that of in



vivo were the % of linalool was 0.0079 with a retention time 11.959 with a area of 1969 in addition to this some 30-

35 other constituents were also detected. (Chromatograms: 1, 2&3).

ENCAPSULATION OF AXILLARY BUDS:

The axillary buds of Lavandula angustifolia measuring 0.5 cms were excised from in vitro raised plants through

direct regeneration and mixed in liquid initiation medium (murashige and skoog’s basal medium) fortified with BAP

(8.88μΜ) and containing Sodium Alginate (SA) and Carboxyl methyl Cellulose (CMC) separately at different

concentrations ranging from 2%, 4%, 6% and 8% to study their effect on encapsulation.

In this present study, it was observed that the complexing ability of Sodium Alginate and Carboxyl methyl Cellulose

varied considerably. Of the two CMC failed to complex with CaCl2 producing only soft and fragile beads in all the

four concentrations tested (Table 2). Further, it was also noticed that axillary buds encapsulated with SA formed

firm, uniform beads and survived better when compared to explants encapsulated with CMC.

Based on the experimental results, Sodium Alginate (SA) was selected for encapsulation of axillary buds in the

present study. The following variables were tested to study the encapsulation (1) Concentration of hydrogel Sodium

Alginate (SA) (2%,4%,6% and 8%), (2) Concentration of complexing agents Calcium Chloride CaCl2 solution

(25mM,50mM , 75mM and 100mM) and (3) Duration of exposure to Calcium Chloride CaCl2 (20 mins, 30mins, 40

mins,50 mins and 60mins) and the results are presented below.

Effect Of Concentration Of Hydrogel Sodium Alginate (Sa)

Further, axillary buds encapsulated in 4% Sodium Alginate produced uniform beads which were firm enough to

transfer with forceps (Table 2). However, axillary buds encapsulated in 6% and 8% produced hard beads (Table 2).

It was observed in the present investigation, that the increase or decrease in concentrations of Sodium Alginate above

or below 4% resulted in poor quality of the beads.

Therefore, 4% Sodium Alginate was found to be the best matrix (hydrogel) for encapsulation of axillary buds.

Effect Of Concentration Of Complexing Agent Calcium Chloride (Cacl2)

In the present study, it was observed that 25mM concentration of Calcium Chloride

(CaCl2) produced very soft synthetic seeds, which were very difficult to handle (Table3).

However, 50mM concentration of CaCl2 resulted in firm, transparent and uniform beads, which were easy to handle

(Table3). Further increase in the concentration of CaCl2 to 75mM and 100mM resulted in opaque and hard beads

(Table3). It was noticed that the increase or decrease in concentration of CaCl2 above or below 50mM resulted in

poor quality of beads.



Therefore, in the present study 50mM CaCl2 was found to be the best complexing agent for encapsulation of in vitro

axillary buds of L. angustifolia.

Effect Of Duration Of Exposure To Calcium Chloride

The axillary buds encapsulated in 4% Sodium Alginate were hardened with 50mM CaCl2 solution at different

duration of time to study optimum exposure to obtain firm and transparent beads.

It was noticed that the hardening of encapsulated beads for 20 minutes and 30 minutes in CaCl2 solution resulted in

very soft beads (Table 4).

However, it was observed that hardening of encapsulated beads for 40 mins. In CaCl2 solution resulted in firm and

strong beads, which were easy to transfer with forceps (Table 4). Further increase in the duration of exposure to 50

mins and 60 mins produced very hard beads (Table 4).

Therefore, in the present study, it was found that 40 mins, exposure in CaCl2 solution was optimum for hardening of

encapsulated in vitro axillary buds of L.anustifolia.

In the present study, it was found that in vitro axillary buds of L.angustifolia can be successfully encapsulated in

4% Sodium Alginate upon complexation with 50mM CaCl2 for 40 mins. To obtain firm, transparent and uniform

synthetic seeds.

REGENERATION OF SNYTHETIC SEEDS

In the present study, it was found that in vitro axillary buds of L. angustifolia by encapsulation in 4% Sodium

Alginate with complexation in 50mM, CaCl2 for 40 mins and was cultured on MSBM supplemented with growth

regulators to study their regeneration capacity (Table 5).

AXILLARY BUD SYNTHETIC SEED

The encapsulated axillary buds of L. angustifolia (Fig.10) were inoculated regenerating medium on MSBM fortified

with BAP (8.88 μΜ) +NAA (2.68 μΜ) in order to retrieve the plantlets.

Germination of synthetic seeds (Fig11.) was observed after days 18 days of culture with 94% of response. After 25

days of culture, shoot initiation with 2–4 leaves (Fig.12) was noticed, which increased to form 2-3 multiple shoots

(Fig.13) after 32 days of culture. Further, elongation and multiplication of shoots (8-10) were noticed after 42 days of

culture (Fig.14). Healthy, elongated shoots (20-25) were observed after 56 days of culture which formed roots on the

same medium after 63 days of culture (Fig.15) the well developed multiple shoots with roots (Fig.16) were subjected

to hardening process (Fig. 17).



It was found in the present investigation that MSBM supplemented with BAP (8.88μΜ) and NAA (2.68 μΜ) was

suitable medium for regeneration of synthetic seed derived from axillary bud explant of L.angustifolia.

In Vitro Conservation and Regeneration

In the present study, the synthetic seed derived from axillary bud explants of L.angustifolia were stored at 40c in

sterile water for 1-6 months to test their viability.

It was noticed that the synthetic seeds stored for 1 month, after exposure to room temperature germinated like normal

seeds after days of culture with % response on MSBM fortified with BAP (8.88μΜ) and (Table 5).

Further, it was observed that the seeds stored for 1 month after exposure to room temperature, germinated like

normal seeds after 2 weeksof culture with 90% response(Table 5,Graph 2 ) on MSBM fortified with

BAP(8.88μΜ)and NAA(2.68 μΜ) seeds stored for 2, 4,and 6 months resulted in retrieval of plants on the same

medium with varying percentage of synthetic seed germination(Table 5). However, synthetic seeds stored for 1-6

months resulted in retrieval of plants with decline in germination percentage when compared to the seeds stored for

1-4 months (Table 5).

In the present study, it was found that synthetic seeds derived from axillary buds explants of L.angustifolia could be

stored for 1-4 months without significant loss of viability. However, 1-6 months old seeds revealed decline in

germination percentage.

DISCUSSION

It was found in the present study, that MS basal medium supplemented with BAP (8.88 μΜ) and NAA (2.68 μΜ)

was the best medium for in vitro axillary bud initiation and shoot multiplication of L. angustifolia. Further, it was

significantly superior than the other growth regulators tested. This finding does not coincide with the findings of

Panizza and Tognoni (1992).

However this agrees with the findings of Miugel and Maria (1996) who have reported that the micropropagation of

L. latifolia is through axillary bud culture on MSBM supplemented with BAP and NAA.The present findings are in

accordance with the findings of Anamaria et al., (1998) and Andrade et al., (1999) and Dias et al., (2002).

Plants of lavender produced in vitro, acclimatized and planted in the field showed good quality of characteristics and

were true-to-type in essential oil composition. Production of plants in vitro could help to counteract the effects of the

decline phenomenon occurring in lavender.



Micropropagation in lavender through axillary buds can be considered as a reliable method for rapid multiplication

of elite clones as well as a potential useful tool for overcoming clonal problems.

In the present investigation, it was found that high percentage of Linalool 0.0079 % percent in the in vivo leaf and

stem of lavender when compared with in vitro leaf and stem (0.004 %).

In the present study 4% Sodium alginate upon complexation with 50mM Calcium Chloride (CaCl2) for 40mins was

found to be best hydrogel and complexing agent for encapsulation of axillary bud. Further, the firm and uniform

beads so obtained resulted in retrieval of plants through in vitro germination of synseeds.

The synthetic seeds of axillary buds can be stored at 4oC in sterile water upto six months.

They germinate and develop into plants when transferred to room temperature on MS basal medium fortified with

BAP (8.88µΜ) and NAA (2.68 µΜ) for Lavandula angustifolia.

REFERENCES

Anamaria Jordan, M. C., Calvo M. C., and Segura, J. 1998. Micropropagation of adult Lavandula dentata plants.

Journal of Horticultural Sciences and Biotechnology, 73(1): 93-96.

Andrade, L.B, Echeverrigaray, S., Fracaro, F., Pauletti G. F. and Rota, L. 1999. The effect of growth regulators on

shoot propagation and rooting of common lavender (Lavandula vera DC). Plant cell, Tissue and Organ Culture,

56: 79-83.

Dias, M.C., Almeida,. R. and Romano A 2002. Rapid clonal multiplication of Lavandula viridis L’He’r through in

vitro axillary shoot proliferation. Plant cell, Tissue and Organ Culture, 68: 99-102.

Dronne, S., Colson, M.., Moja, S. and Faure, O. 1999. Plant regeneration and transient GUS expression in a range of

lavandin (Lavandula x intermedia Emeric ex Loiseleur) cultivars. Plant cell, Tissue and Organ Culture, 55: 193-

198.

Duke, J.A.1985. A hand book of Medicinal Herbs, CRC Press, Inc., Boca Raton, Florida, p.273.

Lis-Balchin, M., Deans, S.G. and Eaglesham,E.(1998). Relationship between the bioactivity and chemical

composition of commercial plant essential oils, Flav. Fragr.j., 13, 98-104.



Lis-Balchin, M. and Hart, S. (1999).Studies on the mode of action of the essential oil of lavender (Lavandula

angustifolia Miller.), Phytother, Res., 13, 540-2.

Lis-Balchin, M. (2002a). Histroy of nomenclature and location of Lavandula species, hybrids and cultivars, in M.

Lis-Balchin (ed.) Lavender: The genus lavandula: Medicinal and Aromatic plants–Industrial Profiles, Taylor and

Francis, London, pp51-6,805.

Malathy Suryanarayanan and Jagadish S. PAI. 1998. Studies in micropropagation of Coleus forskohlii. Journal of

Medicinal and Aromatic plant Sciences, 20: 379-382.

Maria Carmen Calvo and Juan Segura .1989. In vitro propagation of Lavender. Hort Science, 24(2): 375-376.

Mathur, R. J., Ahuja, P. S., Lal, N. and Mathur, A.K. 1989. Propagation of Valleriana wallichii DC. Using

encapsulated apical and axial shoot buds. Plant Science, 60: 111-116.

Melissa Sicurani, Emanuele Piccioni and Alvaro Standardi 2001. Micropropagation and preservation of synthetic

seed in M.26 apple rootstock I: Attempts towards saving labor in the production of adventitious shoot tips suitable

for encapsulation. Plant Cell, Tissue and Organ Culture, 66: 207- 216.

Miguel Carlos Sanchez- Gras and Maria Del Carmen Calvo. 1996. Micropropagation of Lavandula latifolia through

nodal bud culture of matured plants. Plant cell, Tissue and Organ Culture, 45: 259-261.

Panizza, M. and Tognoni, F. 1992. Micropropagation of Lavandin (Lavandula officinalis chaix x Lavandula

latifolia villars c.v. Grosso) In: Bajaj, y.p.s. (ED.), Biotechnology in Agriculture and Forestry: High-tech and

micropropagation 111, springer- verlag Berlin Heidelberg, 19: 295-305.

Panizza, M. and Tognoni, F. 1988. Clonal Propagation, Callus formation and regeneration of Lavandin. Scientia

Horticulturae, 37: 157-163.

Quazi M. H. 1980. In vitro multiplication of Lavandula spp. Annuals of Botany, 45: 361-362.



• Figures (1-9) Initiation and multiplication of shoots from in vitro axillary bud explant of Lavandula angustifolia

• Figures (10-17)

Germination of encapsulated axillary bud of Lavandula angustifolia



TABLE 1 Effect of different concentrations of growth regulators for initiation and multiplication of shoots from in vitro axillary bud of Lavandula angustifolia

ANOVA TABLE (number of shoots/explant)

SV DF SS MSS Fcal ratio Ftabvalue** CD

Treatment 9 3250.05 361.11 74. 45 2.00 4.46 Errors 90 436.70 4.85 Total 99 3686.75

Note: *: Mean no of 10 replication

**: Significance F Value @ 5%le

Basal media

BAP (µM)

BAP (mg/l)

NAA (µM)

Response (%)

No.ofshoots/ explant

X *+ SD

MS 4.44 1.0 2.68 50 20.10 ± 2.02 MS 6.66 1.5 2.68 73 29.50 ± 1.50 MS 8.88 2.0 2.68 91 36.50 ± 2.29 MS 11.11 2.5 2.68 71 28. 30 ± 2.45 MS 13.32 3.0 2.68 66 26.50 ± 2.15

Kinetin (µM) Kinetin ( mg/l) 2,4D(mg/l)

MS 4.64 1.0 2.26 46 18.50 ± 1.74 MS 6.96 1.5 2.26 51 20.40 ± 2.87 MS 9.28 2.0 2.26 71 28.5 0 ± 1.80 MS 11.60 2.5 2.26 51 20.60 ± 1.56 MS 13.92 3.0 2.26 46 18.40 ± 2.10



0

10

20

30

40

50

60

70

80

90

100%

Res

pons

e

1 1.5 2 2.5 3

Growth Regulators (mg/l)

BAP KN

Graph 1: Effect of different concentrations of growth regulators on initiation and multiplication of shoots from in vitro axillary bud explant of Lavandula angustifolia

0

10

20

30

40

50

60

70

80

90

100

Ger

min

atio

n %

1 2 3 4 5 6 7

Duration (months)

Axillary bud

Graph 2: Percentage of germination response of encapsulated axillary buds L.angustifolia



CHROMATOGRAM 1 CHROMATOGRAM 2

CHROMATOGRAM 3



Table 2: Effect of different concentrations of Sodium Alginate and Carboxy Methyl Cellulose for encapsulation of axillary buds of L.angustifolia.

Table 3: Effect of different concentration of Calcium Chloride (CaCl2) for synthetic seed formation of aseptic axillary bud explants of L.angustifolia. Concentration of CaCl2

No. of beads produced

Quality of beads

25mM 30 Soft and transparent

50mM 30 Firm and transparent

75mM 30 Hard

100mM 30 Very hard

Types (w/v) and concentrations % of gel

No. of beads produced

Quality of beads

Sodium alginate

2

4

6

8

30

30

30

30

Soft

Firm

Hard

Hard

Carboxy methyl cellulose

2

4

6

8

30

30

30

30

Soft

Soft

Soft

Soft



Table 4: Effect of duration of exposure to Calcium Chloride for synthetic seed Formation of aseptic axillary bud explants of L.angustifolia.

Table 5: Percentage of germination of synthetic seed of L.angustifolia

Duration mins. No. of beads produced Quality of beads

20 30 Soft

30 30 Soft

40 30 Firm

50 30 Hard

60 30 Very hard

Media Growth regulators BAP (µM)

Growth regulators NAA(µM)

Duration Months

No. of beads stored (40c)

Culture duration

Germination % Axillary buds Lavandula angustifolia

MS 8.88 2.68 0 30 2 weeks 95

MS 8.88 2.68 1 30 2 weeks 90

MS 8.88 2.68 2 30 2 weeks 88

MS 8.88 2.68 3 30 2 weeks 86

MS 8.88 2.68 4 30 2 weeks 84

MS 8.88 2.68 5 30 2 weeks 70

MS 8.88 2.68 6 30 2 weeks 52

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USING IN VITRO TECHINQUES - Centre for … · USING IN VITRO TECHINQUES Leelavathi D ... Effect Of Concentration Of Complexing Agent ... It was noticed that the hardening of encapsulated