Indian Journal of Experimental Biology Vol. 42, January 2004, pp. 106-110

Production of 2-hydroxy-4-methoxybenzaldehyde in roots .of tissue culture raised and acclimatized plants of Decalepis hamiltonii Wight & Am., an endangered shrub endemic to Southern India and evaluation of its performance vis-a-vis

plants from natural habitat

P Giridhar, T Rajasekaran, S Nagarajan" & G A Ravishankar*

Plant Cell Biotechnology Depmtment, Plantation Products, Spices & Flavour Technology Departmenta, Central Food Technological Research Institute, Mysore 570 013, India

Received 18 March 2003; revised 6 October 2003

Axillary buds obtained from field grown plants of D. hamiltonii were used to initiate multiple shoots on Murashige and Skoog's medium (MS) supplemented with 2 mg L-1 6-benzyl aminopurine (BA) and 0.5 mg L- 1 indole-3-acetic acid (lAA). Profuse rooting was achieved when the actively growing shoots were cultured on MS medium supplemented with 1.0 mg r 1

indole-3-butyric acid (IBA). Regenerated plants were grown successfully in the plains, in contrast to wild growth in high altitudes and rocky crevices of hilly regions. Roots of different sizes from one-year-old tissue culture raised field grown plants had the same profile of 2-hydroxy-4-methoxybenzaldehyde as that of wild plants. A maximum of 0.14% and 0.12 % 2-hydroxy-4-methoxybenzaldehyde was produced in roots of one year old tissue culture derived plants and greenhouse grown plants respectively .

Key words: Decalepis, In vitro shoots, Tuberous roots , Flavour compound

Decalepis hamiltonii Wight & Arn., is a monogeneric climbing shrub, native of Deccan peninsula and forest areas of Western and Eastern Ghats of South India. This plant mostly inhabits the mountain and temperate regions in the crevices of rocks and in shady places of high altitudes. The high value aromatic roots have been used as culinary spice, flavouring principle, used as a folk medicine in India as a flatulent reliever, ap­petizer, blood purifier, preservative1 and as a source of bioinsecticide for stored food grains2.-5. The soliga tiibal of Kamataka State and yanadi tribal of Andhra Pradesh State prepare chutney from these roots, which gives ginger like taste. It is used along with food to prevent indigestion, constipation and gas trouble. They also pickle these roots along with lemon juice. Malayas tribe in Kerala uses this root juice as cooling effective drink in summers.

Popular drinks called "Sugandhapala" in Karnataka, "Nannari" in Andhra Pradesh and "Sarsaparilla" in

Abbreviations: 2H4MB: 2-hydroxy 4 methoxy benzaldehyde, IAA : Indole - 3-acetic acid, IBA: Indole-3-butyric acid, BA: 6 benzylaminopurinc, GC: Gas chromatography, GC-MS: Gas chromatography-Mass spectra *Correspondent author: Phone: +91 -0821-516501, Fax: +91-0921-517233, E-mail: pcbt@cscftri.ren.nic.in

Kerala State are made mainly from the highly aromatic roots of Decalepis that are considered as medicinal drinks and have a great demand in summer season as a cooling agent. Mostly the tribals of the area harvest roots during March-May, as it is the main source of their income till the agriculture work resumes 1

• Each root attain 5-10 em in diameter and 8-12 roots grow from each stock. Milky latex is present in the entire plant; a characteristic feature of Asclepiadaceae and a 2-3 year plant gives an average yield of 14-16 kg of roots. Unfortunately adulteration of Hemidesmus indicus for flavour purpose with roots of D. hamiltonii has resulted in its destruction due to ruthless harvesting for decades rendering these plants a place in the endangered lise·5.

Owing to the diverse biological activity of their roots, measures to develop micropropagation protocols for this endangered shrub with high field survival is essential. Our group earlier rerorted the micropropagation protocols for this plant6

- . Another milestone is acclimatization of this plant to plains, which otherwise naturally grows in high altitudes . In this paper we report the production of 2-hydroxy-4-methoxybenzaldehyde in the tubers produced from micropropagated plants grown in plains in comparison to the plants grown in natural habitat.

GIRIDHAR et al.: PRODUCTION OF 2-HYDROXY-4-METHOXYBENZALDEHYDE 107

Materials and Methods Experimental material- Healthy plants of Decalepis

hamiltonii Wight & Arn were collected from the Gumballi forest range located in B.R.Hills, 80 km from Mysore. Axillary buds were excised and washed under running tap water to remove soil and other superficial contamination. The bud and leaf explants were sterilized using 0.1 % (w/v) mercuric chloride for

· five minutes. The explants were cut into 1 em each and cultured on Murashige and Skoog9 (MS medium) agar medium supplemented with 3% sucrose and incubated at 25° ± 2°C with a light intensity of 40 J..Lmol m·2 s· 1 for 16 hr photoperiod using fluorescent lights (Philips India Ltd.) and 60-70% relative humidity.

Initiation of multiple shoots- Axillary buds and leaf explants were cultured on MS medium containing various levels of growth hormones, viz. 6-benzyl­arninopurine [BA (0.5-4.0 mg L-1

)], a-Naphthalene acetic acid [NAA (0.5-2.0 mg L-1

) ], Indole-3-acetic acid [IAA (0.5-2.0 mg L-1

)] in different combinations .

Effect of auxins and cytokinins on the growth of multiple shoots- Apical or axillary buds shoot tips about 0.5-1.0 em long were dissected from field. Five shoot tips were cultured per ja;· containing 40 ml of MS medium supplemented with auxins [NAA & IAA (0.1-0.5 mg L- 1

)] and cytokinin [BA (0.5-2.0 mg C 1l] to evaluate their . effect on shoot growth and multiplication. Explants having one axillary bud measuring 2-3 em were sub-cultured to fresh medium every 30 days for multiplication (Table 1).

Root Induction - Five-week-old shoots were used for the rooting experiments. Shoots longer than 2.0 em were placed on MS medium supplemented with various levels of a-naphthalene acetic acid [NAA (0.25-1.0 mg L-1

)], indole-3-butyric acid [IBA (0.25-2.0 mg L-1

)] and indole-3-acetic acid [IAA (0.25-1.0 mg L-1

)] either in combination or individually for root induction (Table 2).

Hardening of the plants - Rooted plantlets were removed from the culture bottles, washed in running tap water to remove the traces of media and transferred to pots covered with polybags with 60-70% relative humidity. The hardening mixture composed of red soil: sand: farmyard manure (1: 1:1 ). The hardened plants were transferred to the greenhouse, grown for 2 months and then finally transferred to the field. The seedling plants obtained from natural habitat were also transferred to soil as control. The roots were harvested from one-year old plants for further analysis.

Isolation of flavour compound- The roots were grouped into three categories based on their diameter. Group A is < 0.5 em, group B 0.5-1.0 em and group C 1.0-1.5 em diam. The roots (50 g fresh weight,) were cleaned, washed to remove the soil and the central hard woody core separated out. The tubers were dissected into small pieces of 0.5-1.0 em diam and subjected to steam distillation for 5 hr. The steam condensate was extracted with dichloromethane (50 m1 x4). The combined extracts were passed through a funnel containing anhydrous sodium sulphate to remove the water content, concentrated in a flash evaporator and dissolved in lml ethanol and stored in closed vials. Quantification of the flavour compound was determined by gas chromatographic analysis (GC) using flame ionization detection (FID) and GC-MS analysis.

Estimation of flavour compound- Analysis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) which is an isomer of vanillin with a molecular weight of 152 (Fig. 1) was done by spotting the root extracts on TLC plate along with standard (Fluka Chemicals, Switzerland) and run in a solvent system comprising of Hexane: Benzene (1: 1). Rf of spot coinciding with that of standard (2H4MB) (0.47) was eluted in solvent and UV spectrum was measured on a Perkin-Elmer UV-Vis recording spectrophotometer UV -160. Maximum absorption was obtained at 278 nm. Quantitative detection was done by GC-MS and FID. The constituent was identified by matching the mass spectra with GC-MS library user generated mass spectral libraries, and also confirmed by comparison with GC retention time of standard sample. First the standard solution of 2H4MB and root extracts were separated by a gas chromatograph GC-8000 series (Fisons Instruments) using Flame ionization with capillary column, column temperature program was set from · 60°C to 250°C with an incremental rate of 2°C/rnin. The carrier gas was nitrogen with a flow rate of 30 ml/rnin. Detection temperature was 250°C and injection temperature was 100°C. Both standard and root extracted (alcohol) sample (1 J.Ll) was injected separately in gas chromatographic analysis. The retention time of the individual peaks in standard

¢-OH O-CH

3

Fig. ]-Chemical structure of2-hydroxy-4-methoxybenzaldehyde

108 INDIAN J EXP BIOL, JANUARY 2004

sample and root extracted samples were compared and also quantitative percentage calculations was done.

GC-MS method: In order to identify the constituents of 2-hydroxy 4-methoxy benzaldehyde and the concentration of desired compounds, mass spectral analysis was carried out by using GC-MS (Shimadzu), GC-14B coupled with QP 5000 MS system under the following conditions SPB-1 column (Supelco, USA, 30 mm x 0.32 mm, 0.25 p,M film thickness); oven temperature programme, 60°C for 2 min, rising at 2°C/min to 250°C, held for 5 min; injection port temperature 225°C; detector temperature, 250°C; carrier gas helium flow rate 1 ml min ·1. The amount of sample (dissolved in alcohol) injected was lp,l for analysis. GC-MS separation profile of alcohol extract of the root extract indicated the presence of 2H4MB being predominant. The standard and root extract derived ones showed identical compounds during FID and GC-MS analysis. Identification and quantification based on FID and GC-MS analysis respectively.

Statistical analysis - The experiment was repeated twice with 20 replicates each for in vitro shooting , rooting and 5 replicates of roots for flavour compound analysis. The mean (± SE) values of the results were determined. Significant differences between the control and treatments and the results were analyzed using Tukey Multiple Comparison (Software Prism 3.0)

ResultS and Discussion Multiple shoot formation- Shoot bud

development was observed from all the explants in MS medium supplemented with BA (4 mg L'1), BA + NAA (2.0 mg L-1 + 0.5 mg L'1), BA + IAA (2.0 mg L-1 +0.5 mg L'1) and BA + NAA (5.0 mg L-1

+0.5 mg L'1). Axillary bud explants with BA (4 mg L'1) gave good response. The highest number of shoots per flask (18 No.) was observed in MS medium with BA (1.5 mg L'1) + IAA (0.5 mg L'1). However lower concentration of BA also resulted in increased shoot length. (Table 1).

Rooting of in vitro raised shoots-Actively growing shoots rooted at all concentrations of IBA (0.25-2.0 mg L'1). NAA promoted root initiation at low concentration (0.25 mg L-\ IBA gave better rooting response with increasing concentration up to 2.0 mg L-1

•· Profuse rooting was observed on full strength MS medium supplemented with 1.0 mg -L'.1

IBA (Table. 2). The hardened plants were subsequently transferred to greenhouse and later onto the field. The roots raised from tissue culture as well as natural habitat were harvested after one year for analyzing the flavour compound.

Synthesis of the flavour compound at different stages of root growth-Decalepis hamiltonii volatile oil consists of more than 20 constituents, of which the major constituent is the flavour compound, 2-hydroxy-4-methoxybenzaldehyde10. Roots raised from tissue culture derived plants also contained 2-hydroxy-4-methoxybenzaldehyde as the major component but with low quantities at early stages of development. This flavour compound was produced at 0.016% in 0.1-0.5 em diarn tissue culture raised roots whereas 0.092% in normal plant roots. Simultaneously in 0.5-1.0 em diam roots exhibited lot of variation in the concentration of the flavour compound between tissue cultured and naturally grown roots (0.049% and 0.10% respectively). When the roots attained the size of 1.0-1 .5 em diam the content of flavour compound stabilized and it was similar in tissue culture raised as well as naturally grown roots (0.14% and 0.12% respectively) (Table. 3).

Micropropagated plants produced lower quantities of 2-hydroxy-4-methoxybenzaldehyde in early stages of growth. However the content of active principle increased and stabilized after attaining root diameter of 1.0-1.5 em. This may be due to association of the metabolite production to matured tissues and also soil micro flora may have its influence. The high p~r cent of yield in case of earlier reports10 was mainly due to

Table I-Effect of auxins and cytokinins (mg L' 1) on shoot

multiplication of Decalepis lzamiltonii in vitro

Growth regulator No. of shoots/ in MS basal medium explants

BA NAA IAA Control

0 + 0 + 0 0.5 ± 0.08 0.5 + 0.1 + 0 8.0 ± 0.6"

0.5 + 0.5 + 0 8.5 ± 0.9·

1.0 + 0.1 + 0 7.2 ± 0.3" 2.0 + 0.5 + 0 7.1 ± 0.5b

0.5 + 0 + 0.1 9 ± 0.3b

0.5 + 0 + 0.5 12 ± o.8·

1.5 + 0 + 0.5 18 ± 1.2c

2.0 + 0 + 0.5 16 ± 2.0c

Shoot length (em)

0.6 ± 0.2 8.5 ± 0.4b

7.9 ± 0.3b 6.3 ± 0.2c

6.1 ± 0.5c

12.4 ± o.s• 10.4 ± o.2b 11.2 ± 0.4c 8.4 ± 0.6c

P values: • < 0.01, b < 0.05, c < 0.001 compared to controls

GIRIDHAR et al.: PRODUCTION OF 2-HYDROXY -4-METHOXYBENZALDEHYDE 109

Table 2 - Effect of auxins (mg L-1) on in vitro rooting of axillary shoots of

Decalepis hamiltonii

Cone. of auxin in % of ex plants No.of roots/ Root length (em) % of field MS basal medium responded explan ts survival

IAA NAA IBA

0.25 10.0 ± 1.86 1.0 ± 0.29 1.0 ± 0.02 50-60 0.5 1.0

0.25 60.0 ± 4.50 2.91 ± 0.25" 0.88 ± 0.66b 70-80 0.5 60.0 ± 4.70 1.50 ± 0.82" 0.49 ± 0.22. 40-50 1.0

0. 25 0.25 45 ± 3.70 1.11 ± 0.62b 1.7 ± 1.18b 50-60 0 .25 0.5 15 ± 2.5 1 2.0 ± 0.42" 1.0 ± 0.04" 30-40

0.25 25 ± 2.34 1.0 ± 0.25c 0.52 ± 0.13" 60-70 0.5 50± 4.25 2.0 ± 0.38c 0.8 ± 0.16c 60-70 1.0 100 ± 7.80 1.9 ± 0.70b 2.22 ± 0.07c 80-90 2.0 100 ± 6.92 1.6 ± 0.82b 1.20 ± 0.48b 70-80

P values:"< 0.01, b< 0.05, c< 0.001 compared to exp1ants cultured at IAA 0.25 mg r 1

Table 3-Production of 2-hydroxy-4-methoxybenzaldehyde (%) in roots of Decalepis hamiltonii plants

Size (diameter) of the roots) (em)

< 0.5 em 0.5-1 .0 em 1.0- 1.5 em

%of 2 hydroxy-4-methoxy benzaldehyde in tissue

culture) raised field grown roots (dry wt)*

0.016 ± 0.0003 0.049 ± 0.0003"

0.14 ± 0.02"

% of 2 hydroxy-4-methoxy

benzaldehyde in nat4fally

field grown roots (dry wt)*

0.092 ± 0.00 1 0.10 ± 0.004b 0.12 ± 0.002"

P values : "< 0.05 , b < 0.001 compared to flavour compound in roots havi ng diameter < 0.5 em, * retention time = 26.23 and retention indices = 825.93

the nalure of root samples used. There also the compound was extracted by the same hydro distillation method and analyzed by GC. But the tuberous roots were collected from plants grown in natural habitat wherein, tubers collected from only 5-7 years old plants with tuber diameter in the range of 2.5-4.0 em. Obviously in such tubers the % yield of compound will be more. But in the present study the tuberous roots of one year old plants of green house grown normal plant and micropropagated hardened plants were taken for experimental purpose. In such plants the size of the root is definitely less compared to the ones in natural habitat. The reasons may be that the greenhouse raised plants were initially confined to restricted area and with limited supply of nutrients. Normally Decalepis that grows in high altitudes and

hilly regions, where the soil conditions and soil microflora also may influence its growth and metabolites production. The flavour compound 2H4MB per cent varies with the size of the roots thar indicates the direct relationship between the maturity of the root (tuber) and flavour compound. When the tubers attain the size of 1.5 em diam the flavour compound was around 0.12 % and 0.14% in green house grown and tissue cultured plants respectively with a marginal improvement in the tissue cultured plants. Even under field conditions the micro propagated plants showed almost same amount of 2H4MB yields to controls, if such plants allowed to grow in natural habitat , chances are more to get higher yields of the compound in tubers.

The production of the flavour compound in roots of tissue culture raised plants is in equal amounts with that of normal plants may be interesting for the large­scale culti vation and commercial exploitation of this highly endangered shrub in plains.

Acknowledgement The authors are thankful to the Department of

Science and Technology, Government of India for the financial support.

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110 INDIAN J EXP BIOL, JANUARY 2004

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