Introduction

Traditionally, musk for perfume has been obtained from the pods of the male musk deer but only after the sacrifice of the animal. This makes it expensive, rare and unethical. The artificially synthesized musk odorant substances are abundant and cheap but have toxic side effects.  The seeds of Abelmoschus moschatus Medic syn. Hibiscus abelmoschus Linn (commonly known as Mushkdana, Kasturi bhendi) are valued commercially as plant source of musk odorant substance¹. These seeds are a healthier alternative to artificial musk, economical and an ethical substitute for animal musk.

The plant is native to India and is popularly known as Ambrette¹. The seeds find application in Ayurvedic and Unani systems of medicine³. Different parts of this plant are used to treat various disorders of the alimentary tract, heart and skin³.

The distillation of the seeds of Abelmoschus moschatus yield ambrette oil²,  with farnesol  and ambrettolide being its major constituents. The extracts of the plant possess compounds like β-sitosterol, myricetin and their glucosides⁵. The essential oil of ambrette seeds promoted dermal papilla cell growth and therefore, probably, is a promising active ingredient for hair re-growth preparations⁶. Myricetin purified from the aerial parts of A. moschatus lowered plasma glucose levels and increased insulin sensitivity in streptozotocin-induced diabetic rats⁷ thus revealing a potential for antidiabetic drug development⁸. Root extracts of A. moschatus showed larvicidal activity against mosquito larvae⁹. Reports suggest that ambrette oil is comparable to groundnut oil in all the parameters and is suitable for edible use⁴. Its stem bark is reported to yield a jute-like fibre with 78% cellulose¹⁰.

A large number of plants can be produced by micropropagation. Few protocols of micropropagation of this plant have been reported. In this report, we describe the micropropagation of Abelmoschus moschatus, using apical and axillary nodal explants obtained from axenic seedlings grown in vitro. All the experiments were done using ten replicates for each medium and each experiment was repeated thrice.

Materials and Methods

Fresh, germination quality, seeds of Abelmoschus moschatus were collected from various sources across India namely, Naik Krishi Udhyog and Co., Pune; AayurMed Biotech P. Ltd, Mumbai and Gautam Global, Dehra Dun. The surface sterilization method suggested by Maheshwari P et al.¹¹ was modified for these experiments. The seeds were soaked in water for 30mins and washed with dilute liquid detergent (Teepol; Reckitt Benckiser, India) for 5mins, 1% v/v solution of antiseptic (Savlon; Johnson & Johnson Ltd, India) for 60s and 0.1% w/v solution of Mercuric chloride (HgCl2, Suvidhinath Laboratory, India ) for 90s. The seeds were then rinsed thoroughly with sterile double distilled water (x5). Surface sterilized seeds were germinated in half strength Murashige and Skoog’s medium¹² with 1.5% w/v sucrose (pure grade; SRL, India).

The apical and axillary nodal explants of the axenic seedlings were inoculated in MS medium or MS medium supplemented with different concentrations of IAA (Indole-3-acetic acid; Suvidhinath Laboratory, India), NAA (α-Naphthalene acetic acid extra pure; Himedia, India), BAP (N⁶-Benzyladenine extra pure AR; SRL, India) and K (Kinetin pure; Himedia, India) with Sucrose 3% w/v (pure, SRL, India).

Based on the available literature on A. moschatus shoot regeneration, various media were devised to induce shoots namely, MS; MS + 0.55µM NAA + 18µM BAP; MS + 0.55µM NAA + 18µM K; MS + 0.55µM IAA + 18µM K;   MS + 0.55µM IAA + 3µM K;   MS + 0.55µM IAA + 3µM BAP;   MS + 0.55µM IAA; MS + 3µM K; MS + 1µM BAP; MS + 3µM BAP.

The pH of all the media was adjusted to 5.75 using 1N NaOH or 1N HCl. The media were then solidified using 0.9% Agar (Himedia, India), prior to autoclaving at 1.05 Kg/cm² and 121° for 20mins. Explants were inoculated in 10ml of medium in Borosil glass test tubes (150 x 25mm) for seed germination and further for shoot initiation and root induction. The explants were cultured in Plant Tissue Culture laboratory conditions of cool white fluorescent light (40.5 µmol/sq.m/s²) with a photoperiod of 16hrs light and 8hrs dark at 22° ± 2.

The shoots thus obtained in ten days were transferred for root initiation and development in MS + 0.55µM IAA medium. The shoots that developed a dense network of roots were retained in same medium till they attained the height of 8–9cm. The shoots that had just initiated roots or had failed to root in ten days were subcultured in fresh MS + 0.55µM IAA medium The plantlets that had reached the required height were hardened in a sterilized mixture of coarse vermiculite and sand (filled to a height of about 60mm, in clear glass jars of 1L capacity) and wetted with half strength of MS liquid medium. These were maintained for five days in controlled Plant Tissue Culture laboratory conditions. Subsequently they were shifted to room temperature and natural light for next five days for acclimatization. The plantlets were then exposed to non-sterile conditions, though retained in the vermiculite sand mixture for another five days before they were potted in soil.

All quantitative results were tested for significance using ANOVA (1% level of significance) and the comparison of the means was done using the Duncan Multiple Range Test¹⁵. The colour of the leaves obtained in a medium was qualitatively expressed by determining the statistical parameter ‘Mode’ for all thirty replicates of the medium.

Results and Discussion

The axenic seedlings were grown for 15 to 30 days to obtain 3 to 5 nodes per seedling. The shoots were initiated from nodal explants in 1 to 2.57 days in different shooting media (Table 1, Row A; Figure 1-B). The fastest rate of shoot initiation was observed in MS medium.

Within ten days, the shoots showed difference in quality and quantity of foliage. The shoots in MS + 0.55µM IAA medium showed maximum number of leaves but larger leaves were observed in MS + 1µM BAP medium (Table 1, Row B). The colour of the leaves in different media varied from pale yellow to dark green.  The shoots in MS, MS + 0.55µM IAA, MS + 0.55µM IAA + 3µM K and MS + 3µM K media showed fresh dark green leaves. Sporadic instances of multiple shoot formation were observed in MS + 0.55µM NAA + 18µM K; MS + 1µM BAP; MS + 0.55µM IAA + 3µM BAP media with an average of 2.5 shoots per node (Table 1, Row E). Several of these shoots were malformed.

Media that included cytokinins showed callusing at the base of the shoots. This callus was excised from the base before transferring the shoots into rooting medium. However, shoots in some media did not show any callus production during shoot induction such as MS medium and MS + 0.55µM IAA medium (Table 1, Row C; Figure 1-B).

Good shoots with concomitant root initiation were observed in MS and MS medium supplemented with 0.55µM IAA and/or 3µM K, in ten days (Table 1, Row D; Figure 1-C). The number of explants that showed concomitant root initiation was maximum (70%) in MS + 0.55µM IAA medium.

Table 1: Shoot Initiation.

Parameters analyzed during shoot induction in nodal explants of A. moschatus axenic seedlings. The Means ± Standard Deviation were calculated for a sample size of 30 explants. The composition of media a to j are as follows: a. Murashige and Skoog’s medium (MS); b. MS + 0.55µM NAA + 18µM BAP; c. MS + 0.55µM NAA + 18µM K; d. MS + 0.55µM IAA + 18µM K; e. MS + 0.55µM IAA + 3µM BAP; f. MS + 0.55µM IAA + 3µM K; g. MS + 0.55µM IAA; h. MS + 3µM K; i. MS + 1µM BAP; j. MS + 3µM BAP

The shoots from different media were transferred for root initiation and development in MS + 0.55µM IAA medium. The average days required to initiate roots varied from 2.20 ± 0.44 to 26.84 ± 4.38 days (Table 2, Row F; Figure 1-D). The best response was observed in shoots developed in MS + 0.55µM IAA medium. The plantlets that attained a height of 8cm to 9cm with good roots were transferred into vermiculite-sand mixture for hardening. Plantlets regenerated in MS + 0.55µM IAA medium attained the requisite height in the shortest period of time (5.76 ± 1.24 days; Table 2, Row G). Hardening for five days (Table 2, Row H; Figure 1-E) was followed  with  acclimatization for  another  ten  days  and  then the  plantlets  were  transferred to  field conditions.  Plantlets regenerated in MS + 0.55µM IAA medium showed maximum percentage of survival in field conditions (95.83%; Table 2, Row I; Figure 1- F).

Table 2: Root Induction, Survival In Hardening And Field Conditions.

Shoots developed in the media a to j were transferred to MS + 0.55µM IAA medium for root induction. The composition of media a to j are as follows: a. Murashige and Skoog’s medium (MS); b. MS + 0.55µM NAA + 18µM BAP; c. MS + 0.55µM NAA + 18µM K; d. MS + 0.55µM IAA + 18µM K; e. MS + 0.55µM IAA + 3µM BAP; f. MS + 0.55µM IAA + 3µM K; g. MS + 0.55µM IAA; h. MS + 3µM K; i. MS + 1µM BAP; j. MS + 3µM BAP. Their performance in root induction medium was analysed. The rooted shoots were then assessed for their survival in hardening and field conditions.

Figure 1: Micropropagation of Abelmoschus moschatus.   Click here to View figure

A, Seed germination in MS½ medium; B, Shoot induction without callus formation at the base of the explants in MS + 0.55µM IAA medium; C, Concomitant root initiation with shoot induction in MS + 0.55µM IAA medium; D, Root induction in MS + 0.55µM IAA medium; E, Hardening in Vermiculite-sand mixture wetted with MS½ Liq. medium; F, Plantlet in soil.

Nodal explants obtained from axenic seedlings in the above protocol showed rapid shoot initiation in MS medium in one day. The earlier micropropagation studies reported that explants derived from green house grown plants initiated shoots in seven days in MS + 4mg/L BAP + 0.1mg/L NAA medium¹¹  and  cotyledonary nodal explants of A. moschatus initiated shoots in MS + 0.01mg/L Thidiazuran (TDZ) in eight days¹³.

Medium with 1µM TDZ l^-1 was reported to initiate multiple shooting in Hibiscus cannabinus (Kenaf)¹⁴. Multiple shoots in A. moschatus were reported by Renu S. et al.¹³ and Maheshwari P et al.¹¹, using MS + 0.01mg/L Thidiazuran (TDZ) and MS + 4mg/L BAP + 0.1mg/L NAA respectively. Sporadic multiple shoot formation was observed in the current studies in MS + 0.55µM NAA + 18µM K; MS + 1µM BAP and MS + 0.55µM IAA + 3µM BAP media. However, these shoots could not be subcultured.

The present studies also indicated that nodal explants initiated callus at the base of the shoots in the presence of increasing concentrations of cytokinins. Reports in literature indicate that explants of A. moschatus in medium with NAA alone, regenerated shoots accompanied with callus or root initiation¹¹. Renu S. et al. reported that kinetin in the medium promoted callusing in cotyledonary nodal explants of A. moschatus¹³.

Regenerated shoots of A. moschatus were rooted using NAA¹¹ and IBA¹³. IAA has been used by other investigators to initiate roots in tissue culture¹⁶. In the present study, along with shoots regenerated in MS + 0.55µM IAA medium a good and strong network of roots was also induced. Furthermore, it was observed that media supplemented with NAA promoted callus induction at the base of shoots. (Table 1, Row C). Thus, IAA was considered appropriate for root induction.

The plantlets were transferred to field conditions after hardening and acclimatization in a period of 15 days.

MS medium and MS + 0.55µM IAA medium were very good for regeneration of plantlets. However MS + 0.55µM IAA medium could be chosen to be the best medium in this study considering parameters such as the number of leaves in a period of ten days, absence of callus, rate of root induction and root quality. These plantlets could also be transferred for hardening in the least possible time with the best survival in field conditions.

The ambrette seeds are economically very important for their use in perfumery. This technique would enable the production of a large number of plants, rapidly and economically, from a small sample of seeds.

Acknowledgment

We thank Dr. B. L. Jadhav for his encouraging support.

References

1. Bown D., New Encyclopedia of Herbs & Their Uses, Dk Pub, America, 96 (2001)
2.  Fortineau A. D., Journal of Chemical Education., 81(1), 45 (2004).
3. Kirtikar K. R., Basu B. D., and Blatter E., Indian medicinal plants, Pub. Bishen Singh Mahendra Pal Singh, India, 330 (1975).
4. Rao Y. R., Jena K. S., Sahoo D., Rout P. K., Ali Shakir, Journal of the American Oil Chemists’ Society., 82(10), 749 (2005).
5. Nair A. G. R., Nagarajan S., and Subramanian S. S., Current Science., 33, 431 (1964).
6. Komaki R., Fragrance Journal, 33(12), 51 (2005).
7. Liu I. M., Liou S. S., Lan T. W., Hsu F. L., Cheng J. T., Planta Med., 71, 617 (2005)
8. Liu I. M., Tzeng T. F., Liou S. S., Lan T. W., Planta Med., 73, 1054 (2007)
9. Dua V. K., Pandey A. C., Alam M. E., Dash A. P., J. Am. Mosq.Control Assoc., 22, 155 (2006).
10. Duke J. A., and duCellier J. L., CRC Handbook of Alternative Cash Crops, Boca Raton (USA). CRC Press, 4 (1993).
11. Maheshwari P. and Kumar A., Journal of Cell & Tissue Research, 6, 627 (2006).
12. Murashige T. and Skoog F., Physiologia Plantarum, 15, 473 (1962).
13. Renu S. and Anwar S., World Journal of Agricultural Sciences, 4(4), 449 (2008).
14. Srivatanakul M., Park S. H., Sanders J. R., Salas M. G. and Smith R. H., Plant Cell Reports, 19, 1165 (2000).
15. Broota K. D., Experimental Design in Behavioural Research, New Age International, New Delhi, 87 (1989).
16. Indu G. and Kulkarni A. R., Annals of Forestry, 5(1), 50 (1997).