Induction of shoots from in vitro cultured roots of Oldenlandia umbellata L. – a dye-yielding plant

M.S. Shekhawat*, N. Kannan, M. Manokari, J. Revathi

K.M. Centre for Postgraduate Studies, Pondicherry-605 008, India

ABSTRACT:

Shoots were regenerated from the cultured roots of Oldenlandia umbellata L. successfully for mass propagation on Murashige and Skoog (MS) medium. The nodal shoots were cultured on 3.0 mgl^-1 Benzylaminopurine (BAP) to induce nodal meristems. The shoots were multiplied well on MS liquid medium supplemented with 1.0 mgl^-1 BAP + 0.5 mgl^-1 Indole-3 acetic acid (IAA). Shoots were rooted on half strength MS medium supplemented with 2.5 mgl^-1 Indole-3 butyric acid (IBA). The in vitro produced roots were further used as explants to initiate root cultures to get secondary metabolites (anthraquinone derivatives/dye). The root pieces were cultured on half strength MS medium supplemented with IBA. The multiplied roots turned yellow in color within 4-5 weeks. Shoots were regenerated from the roots when the roots cultured on MS medium augmented with 1.0 mgl^-1 BAP. Again these shoots were rooted with help of IBA and the plantlets were hardened in green house.

KEYWORDS: Anthraquinone derivatives, Oldenlandia umbellata L., root culture, shoot regeneration.

INTRODUCTION:

Oldenlandia umbellata (family Rubiaceae) is commonly known as ‘‘Indian madder’’, known to yield a color-fast red dye from its roots, and has been used in traditional medicine since ancient times ^[1]. The root bark, preferably of a two years old plant, when used with a mordant will confer red color to calico, wool, and silk fabrics. It is a low growing plant native to India and commonly found in parts of Coromandel Coast^[^2].

All parts of this plant are used in Siddha as well as in Ayurvedic systems medicines for its styptic properties. A decoction of the roots is used as a febrifuge. Both leaves and roots are also deemed good expectorants, and used for treatment of asthma, bronchitis, and bronchial catarrh^[^3]. A decoction of leaves is used as a rinse to treat poisonous bites^[^4]. ^[5]reported a novel pH indicator dye from this plant.

O. umbellata has been exploited for medicinal and dye extraction purposes due to high market demand^[^6]. As a result, natural stands of O. umbellata are fast disappearing and are threatened with extinction due to indiscriminate collection. The plant grows wild in forests, among other areas, and there is no propagation system available to replenish these stands. ^{[7]successfully} developed in vitro mass propagation protocol to conserve this plant. The normal root cultures were established to produce anthraquinone derivatives by Siva et al.^[8]. But to date, there is no literary evidence on induction of shoots from in vitro root cultures of O. umbellata. Therefore, in present study we investigated here an alternate mode of mass multiplication of shoots through root cultures.

MATERIALS AND METHODS:

The nodal explants of Oldenlandia umbellata were collected from the campus of K.M. Centre for Postgraduate Studies, Pondicherry, India. Explants were surface sterilized under aseptic condition in Laminar air flow hood with 0.1% HgCl₂ (w/v) for 4-5 min followed by 4-5 times washing with autoclaved water. MS medium ^[9] supplemented with 3% sucrose was used for culture initiation. Culture medium was solidified by 0.8% (w/v) Agar (Hi-Media, India). The cultures were maintained at 25±2°C under 12-h photoperiod with light intensity of 40-50 μmol m⁻²s⁻¹ Photosynthetic Photon Flux Density (PPFD) and 60% Relative Humidity (RH).

The nodal segments were inoculated on MS medium supplemented with different concentrations (0.5-5.0 mgl^-1) of BAP and Kinetin (Kn) either alone or in combinations. The shoot cultures were further multiplied by repeated transfer of mother explants and subculture of in vitro produced shoots. The original explants were repeatedly transferred (after harvesting shoots) to fresh MS medium supplemented with 3.0 mgl^-1 BAP and 0.5 mgl^-1 IAA to yield more shoots up to five passages. After 2-3 weeks regenerated shoots isolated from explants were cut into segments with 1-2 nodes (1.0-2.0 cm in length) and subcultured on liquid MS medium containing different concentrations of BAP (0.1-3.0 mgl^-1). Rooting of shoots was attempted on agar-gelled medium. The healthy shoots were excised, separated and transferred individually to half strength MS medium + 0.1% activated charcoal with various concentrations (0.5-5.0 mgl^-1) of IBA.

The in vitro regenerated roots were used as explants to establish root cultures for anthraquinone derivatives (dye) production. The root segments were cultured on half strength of MS medium + 0.1% activated charcoal with various concentrations (0.5-5.0 mgl^-1) of IBA for root multiplication. For increased dye production and shoot induction the roots were further cultured on full strength MS medium supplemented with 0.1 to 3.0 mgl^-1 BAP. These shoots were next rooted on half strength MS medium with 2.5 mgl-1 IBA. Rooted plantlets (3-4 cm in length) were hardened in green house. The plantlets were kept in the green house for 4-5 days in capped glass bottles containing autoclaved soilrite moistened with 1/4 strength MS salts. The plantlets were gradually exposed to green house conditions by loosening and removing the caps. The hardened the plantlets were transferred to the pots containing sand, garden soil, and organic manure (2:2:1) and successful hardened plantlets of O. umbellata were transferred to the nursery.

All the experiments were repeated thrice with 15 replicates for each treatment. The results are expressed as mean ± SD of three experiments.

RESULTS and DISCUSSION:

We reported micropropagation protocol for O. umbellate in our previous publication^[7] which we have briefly described here to make continuity with our present investigation and after that we will discussing our new findings in this report. Fresh shoot segments of length 2 to 3 cm with 1-2 nodes were found to be most suitable for culture initiation. A 100% bud break occurred after 15 days of inoculation on MS semisolid medium supplemented with 3.0 mgl^-1 BAP, and maximum of 13.9±0.23 shoots were obtained. This rate of shoot regeneration is very high as compared to previous reports^[^10].

Shoots initiated from activation of axillary meristem were further amplified by repeated transfer of mother explant^[^{11, 12]} or subculturing of in vitro rejuvenated shoots. Repeated transfer was carried out on MS semisolid medium supplemented with 3.0 mgl^-1 of BAP and 0.5 mgl^-1 IAA. On this medium 43-45 shoots were obtained after 15-20 days. This medium was supportive for shoot development up to fourth culture cycle. Multiplication of cultures was best achieved by subculturing shoots on MS liquid medium supplemented with 1.0 mgl^-1 of BAP + 0.5 mgl^-1 IAA for mass multiplication of shoots. It was also observed that culture medium containing more than the average cytokinin, number and length of shoots was reduced^[^{13, 14]}. In vitro rooting of individual shoots was achieved with half-strength semisolid MS medium containing 2.5 mgl^-1 of IBA + 0.1% activated charcoal. About 11-12 roots of length 2-3 cm were obtained after 3-4 weeks of inoculation^[^7].

Experiments were set-up to initiate and enhance in vitro synthesis of anthraquinone derivatives (dye) in root cultures as described by^[^8] and^[15]. The rate of normal root multiplication was quite satisfactory on half strength MS medium with 2.5 mgl^-1 IBA (Fig. 2A and B). Therefore, the roots were transferred on MS medium containing BAP for coloration of roots (dye synthesis). In this course of experimentation we found shoots primordia developed from the roots (Fig. 1A and B). These were further multiplied and elongated ((Fig. 1C and D)) on same media combination within four weeks (Fig. 2C).

The alternate method developed in present investigation is significantly better than the method already described as above and published ^[7]. The number of shoots per culture was more (76.2 shoots per culture) as compared to previous method (Table). The development and elongation of shoots is also better than the earlier report. Above all the hormonal requirement is also low as compared to^[^10] and^[7] findings. Only half strength MS medium is sufficient to get good amount of roots. The maximum number of shoots was regenerated when the multiplied root pieces were sub cultured on MS medium augmented with 1.0 mgl^-1 BAP (Fig 2D). Present method is cheaper than the old method because once the cultures conditions were standardized the sucrose and agar could be replaced by cheaper alternatives, like sugar cubes at the place of sucrose and industrial grade agar at the place of analytical grade agar-agar.

Table: Effects of Cytokinins (BAP and Kn) concentrations on multiple shoots formation from roots on MS Medium after four weeks.

BAP (mgl^-1)	Kn (mgl^-1)	Shoot Number ± SD	Shoot Length (cm) ± SD
0.1	-	44.7 ± 0.24	2.2 ± 0.56
0.5	-	62.9 ± 0.24	3.4 ± 0.33
1.0	-	76.2 ± 0.17	3.7 ± 0.72
1.5	-	62.6 ± 0.93	3.2 ± 0.23
2.0	-	54.6 ± 0.76	2.8 ± 0.87
2.5	-	39.7 ± 0.30	2.4 ± 0.34
3.0	-	22.6 ± 0.56	2.3 ± 0.82
-	0.1	27.4 ± 0.23	2.8 ± 0.83
-	0.5	37.8 ± 0.65	3.4 ± 0.44
-	1.0	54.1 ± 0.94	3.3 ± 0.65
-	1.5	42.4 ± 0.48	3.7 ± 0.21
-	2.0	34.7 ± 0.19	2. 9 ± 0.11
-	2.5	21.5 ± 0.47	2.3 ± 0.62
-	3.0	13.5 ± 0.91	2.7 ± 0.32
0.1	0.1	43.6 ± 0.34	2.2 ± 0.29
0.5	0.5	49.9 ± 0.85	3.1 ± 0.30
1.0	1.0	33.3 ± 0.49	3.1 ± 0.32
1.5	1.5	24.8 ± 0.33	3.4 ± 0.65
2.0	2.0	17.3 ± 0.24	2.8 ± 0.55
2.5	2.5	12.3 ± 0.64	2.1 ± 0.47
3.0	3.0	10.7 ± 0.82	2.7 ± 0.33

Note: The observations were taken after every twenty days of inoculation. The experiments were repeated trice with fifteen replicates per treatment.

Activated charcoal is said to promote in vitro root initiation as well as multiplication in low concentration as it provides darkness and adsorbs PGRs but it was not effective in shoot induction from in vitro cultured roots. Roots could be multiplied fast with activated charcoal but it was ineffective for shoot induction and elongation. Diffused light (10-20 μmolm^-2s^-1 PPFD) also favored in vitro root induction and multiplication but more light intensity (30 to 50 μmolm^-2s^-1 PPFD) was must for shoots formation. IBA is more effective than IAA, NAA and NOA in promoting rooting of a wide variety of plants, and it is used commercially for rooting of many plant species worldwide^{[16, 14]}. Similar results were found in case of rooting of shoots regenerated from the cultured roots as previously reported by us. The plantlets were hardened in green house and transferred from bottles to pots containing sand, garden soil, and organic manure. This is the first report on in vitro propagation of O. umbellata at mass scale from cultured roots. The in vitro methods developed and described in this paper offer an efficient way of mass propagation of this important plant species.

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Received on 15.12.2012

Modified on 23.12.2012

Accepted on 25.12.2012

Research Journal of Pharmacognosy and Phytochemistry. 5(1): January–February 2013, 22-25