Journal of Herbs, Spices & Medicinal Plants, Vol. 14(1–2) 2008Available online at http://www.haworthpress.com© 2008 by The Haworth Press. All rights reserved.

doi:10.1080/10496470802341185 29

WHSM1049-64751540-3580Journal of Herbs, Spices & Medicinal Plants, Vol. 14, No. 1, August 2008: pp. 1–15Journal of Herbs, Spices & Medicinal Plants

Effects of Growth Regulator Application on Growth, Flower, Oil Yield, and Quality

of Clary Sage (Salvia sclarea L.)Singh et al.Journal of Herbs, Spices & Medicinal Plants Virendra Singh

Ruchi SoodKulasekaran Ramesh

Bikram Singh

ABSTRACT. The effects of growth regulator applications (kinetin, IAAand Paclobutrazol) on field grown Salvia sclarea L. were studied during2005 to 2006 at the Institute of Himalayan Bioresource Technology,Palampur, India. Growth was monitored by measuring plant height, can-opy spread, leaf number, primary branches, and yield characteristics(inflorescence length and flower and oil yield). Quality was evaluated.Maximum height and leaf numbers were observed after application ofkinetin 10 μL L−1 and IAA 50 μL L−1, respectively. Maximum flower andoil yield were observed after application with 40 μL L−1 paclobutrazol(PBZ) application. Application of 80 μL L−1 paclobutrazol applicationincreased the linalool-linalyl acetate content of the plant about 12%higher than the untreated control.

Natural Plant Products Division, Institute of Himalayan Bioresource Technology,Palampur 176061 (HP) IHBT Publication No. 0695.

Address correspondence to: Dr. Virendra Singh, Scientist, Natural Plant ProductsDivision, Institute of Himalayan Bioresource Technology, Palampur 176061 (HP),India.

National Bioresource Development Board, Department of Biotechnology, Govt.of India has funded this study. Authors are also thankful to Dr. P.S. Ahuja, Directorof the Institute, for his constant encouragement and valuable suggestions.

Received: January 4, 2007.

30 JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS

KEYWORDS. Aromatic plant, essential oil, IAA, kinetin, linalool, linalylacetate, medicinal plant, paclobutrazol

INTRODUCTION

Clary sage (Salvia sclarea L., family Lamiaceae), is a xerophytic, bien-nial plant native to southern Europe (4). Although typically endemic tothe Mediterranean basin to the Atlantic Ocean, the plant is one of the mostcultivated aromatic plants, especially in France, Bulgaria, West China(14), Central Europe, England, Morocco, and the United States, and isused worldwide as a source as essential oil and other compounds derivedfrom different parts of the plant (13). The plant has diverse biologicalactivities manifested by the different components of the essential oil thatallow for the many medicinal and pharmaceutical applications.

The whole plant, but particularly the inflorescence, possesses a strongaromatic scent, the essential oil, characterized by a fresh floral and herba-ceous aroma, is used in high-grade perfumes, and the essential oil constitu-ent sclareol is used as base material for the industrial synthsis of the highlyvalued ambergris odorants. The chemical composition of the essential oilfrom clary sage is almost exclusively determined by the geographical habi-tat of the plant, whether wild or cultivated (2,9,19,22,25), although a fewdifferences in the oil occur under harvest and cultivation conditions (9).

The objective of this study was to evaluate the effect of foliar applica-tion of growth regulators (kinetin, IAA, and PBZ) on the developmentand essential oil of clary sage growing in the mid-hill region of HimachalPradesh, India.

MATERIALS AND METHODS

Plant Material

Clary sage, (Salvia sclarea L., family Lamiaceae) plants grown fromseed were used in this study. The seeds were planted in a nursery onSeptember 30, 2005, and the plants were transplanted to an experimentalfield located at the Division of Natural Plant Products, Institute ofHimalayan Bioresource Technology, CSIR, located at Palampur (32°06′N, 76°33′ E; 1,300 meters above mean sea level), India on January 16,2006. The planting (plot size, 3.6 m × 2.75 m) was arranged in a randomized,

Singh et al. 31

complete block design with three replications. Locally available farmyardmanure was applied at 20 MT ha−1. Plants were maintained through irri-gation and pest control as needed, following regular growing practices forcultivated clary sage.

Experimental

To determine the effect of growth regulators on clary sage, seven treat-ments were tested, kinetin at 5 and 10 μL L−1 indole acetic acid (IAA) at50 and 100 μL L−1 paclobutrazol (PBZ) (C15H20ClN3O, a triazole-typeplant growth retardant) at 40 and 80 μL L−1, and control (no growth regu-lator). The growth regulators were applied as foliar applications (spray)two times, 15 days apart, before flowering.

The plants were harvested on June 14, 2006 (149 days after planting),by randomly slecing five plants from each plot. Observations were madeon plant height (measured from the soil surface to the terminal shootapex), canopy spread, number of leaves, and number of primary branches.

Essential Oil

The essential oils were extracted from 1 kg of the flowers by hydrodistilla-tion in Clevenger-type apparatus for 3 hours and dried over anhydroussodium sulfate as described by Mastelic et al. (16). The oils were analyzedusing a gas chromatography-mass spectrophotometry (GC-MS) system(Shimadzu model QP 2010, Shimadzu, Kyoto, Japan) equipped with a Carbo-wax 20M using an injection temperature of 220oC, an initial oven tempera-ture of 70oC programmed to hold for 4 minutes, rise from 70 to 220oC at 4oCminutes−1, and hold at 220oC for 5 minutes. The ion source temperature was200oC, and the interface temperature was 220oC. The split ratio was 1:50.

Oil constituents were identified by matching compound mass spectra withcompounds in mass spectrometry libraries and by matching retention indiceson the carbowax column with published data (1,12,17,23). Kováts (reten-tion) indices (18) were calculated by comparing gas chromatograms withhomologues series of n-alkanes (C8–C32) (Sigma-Aldrich, St. Louis, MO).

Data was analyzed using standard statistical methods.

RESULTS AND DISCUSSION

Growth, as measured by plant height, canopy spread, number of leaves,and numbers of primary branches, were significantly influenced by

32 JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS

growth regulators (Table 1). Taller plants, as compared with the control,were observed with the application of kinetin at 10 μL L−1. Similar heightincreases were achieved by plants treated with 50 μL L−1 IAA, and with40 and 80 μL L−1 PBZ. The IAA treatment at 100 μL L−1 reduced plantheights as compared with the lower concentration tested, suggesting thecritical limit for treatment with IAA to be at or near 50 μL L−1. The low-level IAA application yielded maximum values for caopy spread (E–W)(60.3 cm), number of leaves (60.6), and number of primary branches(3.9). The 40 μL L−1 PBZ application yielded minimal values for the cropcanopy (N–S: 50.3 cm), number of leaves (35.3), and number of primarybranches (2.3). The crop canopy (N–S) was maximum for the control(65.2 cm), and the maximum canopy spread was with IAA at the 50 μL L−1

level, possibly owing to higher number of leaves.Inflorescence length was unaffected by growth regulators, although

significant differences in flower yield, oil content, and oil yield wereobserved. Maximum flower and oil yield were rcorded with the 40 μL•L−1

PBZ treatment. In contrast, flower yield was drastically reduced withkinetin at 5 μL L−1and IAA at 100 μL L−1 as compared with the control.Similarly, PBZ appliction induced the highest oil content (25 %; Table 2).PBZ application has been shown to increase chlorophyll a and b contentand photosynthetic efficiency, but delay physiological maturity in pota-toes (26). A delay in maturity may explain the observed increase in floweryield in clary sage. Furthermore, the reduction in vegetative growth fol-lowing PBZ treatment may have caused an altering of relative sink

TABLE 1. Effect of growth regulators on growth of clary sage

Treatment Plant height (cm)

Canopy spread (cm) Leaves (No.)

Primary branches

(No.)E–W N–S

Control 105.1 45.6 65.2 44.9 2.8Kinetin, 5 μL L−1 106.3 55.4 56.6 41.3 2.8Kinetin, 10 μL L−1 115.7 58.0 60.0 44.8 3.0IAA 50 μL L−1 112.7 60.3 58.1 60.6 3.9IAA 100 μL L−1 108.1 53.4 50.7 39.3 3.0PBZ 40 μL L−1 114.0 46.6 50.3 35.3 2.3PBZ 80 μL L−1 113.3 47.8 50.3 50.1 3.4CD (p = 0.05) 6.03 8.06 6.84 12.41 0.86SEM 1.79 3.01 3.17 1.67 0.15

IAA = indole acetic acid; PBZ = paclobutrazol.

Singh et al. 33

strengths within the plant and indirectly enabled a greater partition of theassimilates to reproductive growth, flower bud formation, fruit formation,and fruit growth (7).

Maximum linalool concentration (39.44 % of total oil) was recorded inthe untreated control plants, while maximum linalyl acetate concentration(31.83 % of total oil) was obtained with 80 μL•L−1 PBZ application(Table 3). High levels of linalool and linalyl acetate are required for goodquality oil (24). Although Pešic and Bankovic (20) have reported thatlinalool and linalyl acetate contribute 86.07 percent of the essential oil inclary sage, in our study the linalool and linalyl acetate was only 55.08 to62.79 of the essential oil, possibly due to the selected cultivar. Althoughthe 80 μL L−1 PBZ treatment reduced the yield of linalool as comparedwith the control, the combined linalool and linalyl acetate yield was about12% higher than the control, indicating the PBZ treated plants produced abetter quality of the essential oil. These results are contradictory to thosereported by Mastelic and I. Jerkovic (15). Inflorescences at full floweringstage, however, presented higher linalool, α-terpineol, and germacrene Dbut a lower content in linalyl acetate that conforms to the findings ofCarruba et al. (4).

The role of PBZ as an anti-gibberellin agent is considered sufficient forthe induction of higher yield, improved quality (3,11,21), and increasedabiotic stress resistance in plants (5). Proof about the ways in which thisplant growth regulator changes biochemical plant characteristics, how-ever, is scarce (7). Active PBZ that reaches the subapical meristems

TABLE 2. Effect of growth regulators on flower and oil yield of clary sage

Treatments Inflorescence length (cm)

Flower yield (q/ha)

Oil content (%)

Oil yield (kg/ha)

Control 58.9 31.1 0.19 5.9Kinetin, 5 μL L−1 60.2 16.2 0.21 3.4Kinetin, 10 μL L−1 61.8 33.9 0.19 6.3IAA 50 μL L−1 63.9 36.7 0.18 6.8IAA 100 μL L−1 57.8 16.5 0.20 3.2PBZ 40 μL L−1 61.1 38.6 0.25 9.6PBZ 80 μL L−1 60.0 27.3 0.25 6.6CD (p = 0.05) NS 5.74 0.03 1.33SEM 0.48 1.85 0.01 0.75

IAA = indole acetic acid; PBZ = paclobutrazol.

34

TA

BLE

3. E

ffect

of g

row

th r

egul

ator

s on

oil

qual

ity o

f cla

ry s

age

Com

poun

dsC

ontr

olK

inet

in 5

μL

L−1K

inet

in 1

0 μL

L−1

IAA

50

μL L

−1IA

A 1

00 μ

L L−1

PB

Z 4

0 μL

L−1

PB

Z 8

0 μL

L−1

Mon

oter

pene

s (%

tota

l oil)

Myr

cene

2.07

1.97

2.07

1.99

1.81

2.06

1.61

Ses

quite

rpen

es (

% to

tal o

il)β-

Car

yoph

ylle

ne1.

651.

721.

991.

441.

341.

671.

61

Alc

ohol

s (%

tota

l oil)

Lina

lool

39.4

434

.32

33.0

936

.90

36.5

436

.44

30.9

6α-

Ter

pine

ol17

.17

15.2

315

.08

15.3

214

.12

15.6

813

.19

Ner

ol2.

112.

051.

801.

991.

891.

991.

67G

eran

iol

6.06

5.99

5.19

5.78

5.43

5.75

5.07

Ace

tate

s (%

tota

l oil)

Lina

lyl a

ceta

te15

.64

22.9

823

.97

21.3

124

.64

20.3

531

.83

Ner

yl a

ceta

te3.

243.

153.

183.

152.

983.

182.

62G

eran

yl a

ceta

te6.

596.

376.

426.

335.

836.

435.

23T

otal

93.9

793

.78

92.7

994

.21

94.5

893

.55

93.7

9

IAA

= in

dole

ace

tic a

cid;

PB

Z =

pac

lobu

traz

ol.

Singh et al. 35

inhibits gibberellin production by inhibiting the oxidation of kaurene intokaurenoic acid, a cytochrome P450 catalyzed reaction occurring inmicrosomes (11).

By inhibiting gibberellin biosynthesis, PBZ may slow growth (8) whilemaintaining photosynthetic rates (28) and thus increase carbohydrate con-tent (27–29). Since gibberellins share a biosynthetic pathway with sec-ondary metabolites (10) and PBZ inhibits the synthesis later in the chainof reactions (8), a possible outcome is to increase the availability of pho-tosynthates to be allocated to the production of secondary metabolites.Hence, PBZ may increase secondary metabolism through effects onresource allocation patterns similar to those caused by limited nutrientsupply (6). PBZ blocks three separate steps in the terpenoid pathway. Thiscould account for the increased linalool and linalyl acetate content due toPBZ application.

REFERENCES

1. Adams, R.P. 1989. Identification of Essential Oils by Ion Trap Mass Spectroscopy.San Diego Press, New York.

2. Commission of European Communities. 1990. Official Journal of the EuropeanCommunities. Annex No. C-146 A.

3. Basiouny, F. 1994. Effects of paclobutrazol, gibberellic acid, and ethephon onyield and quality of muscadine grapes. Phyton (Argentina)56:1–6.

4. Carruba, A., R. Torre, R. Piccaglia, and M. Marotti. 2002. Characterization of anItalian biotype of clary sage (Salvia sclarea L.) grown in a semi-arid Mediterraneanenvironment. Flav Fragr J. 17(3):191–194.

5. Chaney, W.R. 2005. A paclobutrazol treatment can leave a tree more stresstolerant. Golfdom:(February issue, 2005).

6. Chorbadjian, R.A., and D.A. Herms. 2006. Effect of plant growth regulatorpaclobutrazol and fertilization on paper birch and Austrian pine resistance to folivores.Accessed on December 7, 2007, at http://hdl.handle.net/1811/6175.

7. Christov, C., I. Tsvetkov, and V. Kovachev. 1995. Use of paclobutrazol to controlvegetative growth and improve fruiting efficiency of grapevines (Vitis vinifera L.). Bulg.J. Plant Physiol. 21(4):64–71.

8. Coolbaugh, R.C., and R. Hamilton. 1976. Inhibition of ent-kaurene oxidation andgrowth by alpha-cyclopropyl-alpha-(p-methoxyphenyl)-5-pyrimidine methyl alcohol.Plant Physiol. 57(2):245–248.

9. Dzumayev, K.K., L.A. Tsibulskaya, I.G. Zenkevich, K.G. Tkachenko, and I.F.Satzyperova. 1995. Essential oils from Salvia sclarea L. produced from plants grown insouthern Uzbekistan. J. Essent. Oil Res. 7:579–604.

10. Hanover, J.W. 1975. Physiology of tree resistance to insects. Annu. Rev. Entomol.20:75–95.

36 JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS

11. Hedden, P., and J. Graebe. 1985. Inhibition of gibberellin biosynthesis bypaclobutrazol in cell free homogenates of Cucurbita maxima endosperm and Malus pum-ila embryos. J. Plant Growth Regul. 4:111–122.

12. Jennings, W., and T. Shibamoto. 1988. Qualitative Analysis of Flavor and Fra-grance Volatiles by Glass Capillary Gas Chromatograph. Academic Press, New York.

13. Lawrence, B. M. 1992. Chemical components of Labiate oils and their exploita-tion. In: T. Reynolds and R.M. Harley, eds. Advances in Labiate Science. BaloghScientific Books, Champaign, IL. pp. 399–436.

14. Leung, A.Y., and S. Foster. 1996. Encyclopedia of Common Natural IngredientsUsed in Food, Drugs and Cosmetics, 2nd ed. Wiley, New York. pp. 173–175.

15. Mastelc, J., and I. Jerkovc. 2003. Application of co-distillation with superheatedpentane vapour to the isolation of unstable essential oils. Flav. Fragr. J. 18:521–526.

16. Mastelic, J., M. Milorš, D. Kuštrak, and A. Radonic. 1998. The essential oil andglycosidically bound volatile compounds of Calamintha nepea (L.) Savi. Croatica Chem.71:147–154.

17. McLafferty, F.W. 1989. Registry of Mass Spectral Data, 5th ed. John Wiley andSons, New York.

18. McNaught, A.D., and A. Wilkinson (compilers). 1997. IUPAC Compendium ofChemical Terminology,2nd ed. International Union of Pure and Applied Chemistry,Research Triangle Park, NC.

19. Peana, M.D.L., C.J. Moretti, and C. Juliano. 1999. Chemical composition and anti-microbial action of the essential oils from Salvia desoleana Atzei & Picci and SalviaSclarea L. Planta Med. 65:752–754.

20. Pešic, P.Z., and V.M. Bankovic. 2003. Investigation on the essential oil of culti-vated Salvia sclarea L. Flav. Fragr. J. 18(3):228–230.

21. Reynolds, A., and D. Wardle. 1990.. Vegetative growth suppression bypaclobutrazol in greenhouse-grown “Pinot noir” grapevines. HortScience 25:1250–1254.

22. Souleles, C., and N. Argyriadon. 1997. Constituents of the essential oil of Salviasclarea growing wild in Greece. Int. J. Pharmacog. 35:218–220.

23. Stein, S.E. 1990. Mass Spectral Data Base and Software, Ver. 3.02. National Instituteof Standards and Technology, United States Department of Commerce, Washington, DC.

24. Svoboda, K.P., G. Ruzickova, and J.B. Hampson. 2001. Bioactivity of essentialoils and their components—Clary sage (Salvia sclarea L.), Part 5. Aroma Res. 2(1):84–89.

25. Torres, M.E., A. Velasco-Negueruela, M.J. Perez-Alonso, and M.G. Pinilla. 1997.Volatile constituents of two Salvia species grown wild in Spain. J. Essent. Oil Res. 9:27–33.

26. Tsegaw, T. 2006. Response of Potato to Paclobutrazol and Manipulation ofReproductive Growth under Tropical Conditions [Ph.D. thesis]. University of Pretoria,Pretoria, Gauteng, South Africa

27. Wang, S.Y., G.L. Steffens, and M. Faust. 1986. Effect of paclobutrazol on accu-mulation of carbohydrates in apple wood. HortScience 21:1419–1421.

28. Wieland, W.F., and R.L. Wample. 1985. Effects of paclobutrazol on growth, pho-tosynthesis and carbohydrate content of ‘Delicious’ apples. Sci. Horticult. 26:139–147.

29. Wood, B.W. 1984. Influence of paclobutrazol on selected growth and chemicalcharacteristics of young pecan seedlings. HortScience.19:837–839.