INTRODUCTION:

It may sound an exaggeration of facts, but there may hardly be any plant, which may not be having medicinal or nutraceutical value. Many plants, which have not been identified as yet through pharmacology, folk medicine, homoeopathy and ethnopharmacology, are being investigated for their medicinal usage and may be proved so in due course of time. Commensurate with this the investigation of biotechnology or to be precise, plant tissue culture for accelerating clonal multiplication of desired clones and strains of medicinal plants through somatic embryogenesis. In genetic improvement schemes, multiplication of elite materials by somatic embryogenesis prevents genetic recombination and the need for long, expensive conventional selection cycles. Chrysanthemum is a cosmopolitan genus, comprising about 300 species of herbs and under shrubs. Several species of Chrysanthemum are ornamentals grown in gardens for their large, showy, multicoloured flowers and are also an important cut flower crop. Chrysanthemum is a mild-acting medicine possessing anti-microbial and anti-viral properties and shows the mildest anti-oxidation activity.

There are various reports on its components such as chlorogenic acid, flavonoids and pentacyclic triterpenes, its clinical applications, its anti-HIV, anti-tumor and anti-mutagenic activities (Chen et al., 2005). Its flowers yield an important insecticide, i.e. the pyrethrins. Pyrethrins, widely used as natural insecticides, offer all the advantages of chemical compounds, that is, rapidity of action against a broad range of insects, and rapid biodegradability (Hitmi et al., 2000).

Even though many reports are available on in vitro propagation, the protocols are complicated. Here, we report a very simple economical, rapidly multiplying and highly reproducible protocol for somatic embryogenesis of Chrysanthemum through suspension culture.

MATERIALS AND METHODS:

The leaves and petals were collected from the green house (Figure1) of the Department of Biochemistry, Biotechnology and Bioinformatics. The explants were washed thoroughly under running tap water for 30 min, followed by 0.5% bavistin for 15 minutes. Bavistin treated explants were washed with sterile distilled water, treated with 5% Tween 20 for 5 min, and washed repeatedly with sterile distilled water. The explants were then surface sterilized with 0.5% mercuric chloride for 2 min and washed with sterile distilled water for 3 times each under the laminar airflow followed by 70% ethanol for 1 min. After washing in sterile distilled water, the explants were inoculated aseptically in MS medium (Murashige and Skoog, 1962) containing 30 g/L sucrose and gelled with 8 g/L diffco bacto agar.

Figure.1 Chrysanthemum Explant

Callus induction and regeneration from Callus:

MS medium supplemented with the varying concentration of the auxin, 2,4-D was used for callus induction studies. The effect of different concentration of the auxin, 2,4-D on callus induction was studied on two explants namely, leaf and petals. All the calli obtained were subculture after 30 days and the friable callus obtained from best concentration were transferred to MS medium supplemented with 1.0 mg/L BAP for regeneration. A photoperiod of 16/8 h light and dark was maintained.

Somatic Embryogenesis in Suspension Culture:

The calli were maintained in MS medium supplemented with 1.0 mg/L BAP for a month and then were transferred to MS liquid medium supplemented with 1.0 mg/L BAP. A photoperiod of 16/8 h light and dark and the cultures were incubated in the shaker at 75-80 rpm.

Shoot Induction, Elongations and Rooting:

The somatic embryos were subcultured after 20 day and were transferred to the following medium for regeneration. In 10 replicates with 3 explants in each replicate were inoculated. Thirty day old regenerated plantlets were transferred to MS medium supplemented with 0.1 mg/L BAP + 2.0 mg/L KIN for elongation. A photoperiod of 16/8 h light and dark was maintained. After 30 days the regenerated plants were transferred in to MS0 for rooting.

Hardening:

The individual rooted plants were carefully taken out, washed free of agar, and transferred to plastic cups filled with sterile vermiculite and maintained in rectangular glass box inside the growth room under high humidity for initial establishment. After 3- 4 days, they were again hardened in the mist chamber for one week for further growth and establishment.

Statistical Analysis:

The data generated from the various experiments were subjected to statistical analysis by using the statistical software AGRES, in completely randomized design (CRD). Percentage values were transformed to arcsine values before statistical analysis, wherever necessary. Each experiment had 10 replicates with three explants each.

RESULT AND DISCUSSION:

Callus Culture:

Effect of 2,4-D Concentration on Callus Induction and Proliferation:

Effect of four different concentrations of 2,4-D (0.5, 1.0, 1.5, and 2.0 mg/L) on callus initiation and callus mass were assessed in leaf and petal explants. Callus initiation and proliferation was observed at weekly intervals. The percentage of callus initiated was recorded 4 weeks after inoculation. The increase in mass as gain in weight was recorded as the proliferation rate of callus after 6 weeks of inoculation.

Effect of 2,4-D Concentration on Callus Induction:

The effect of 2,4-D on callus induction in leaf and petal is summarized in Table1. A significant difference is observed between treatments. A 100% callus induction (Figure 2) was observed for leaf explants in T3 (1.5 mg/L 2,4-D) followed by T4, T2 and T1. The callus induction on MS medium supplemented with 2.0 mg/L 2,4-D (T4) was 81% and 72% in T2 while the callus response was lowest (20%) in T1 (0.5 mg/L 2,4-D). A high callus response of 100% was achieved in MS medium supplemented with 2.0 mg/L 2,4-D (T4) for petal explants. The callus formation was 80% in T3 (1.5 mg/L 2,4-D) and 48% in T2 (1.0 mg/L 2,4-D). The callus induction was found to be lowest T1 (24%) for petal explants.

Figure.2 Green Callus

Obukosia et al., 2004 studied the effect of growth regulators on culture response in Chrysanthemum, the results indicated that MS medium supplemented with 2.0 mg/L 2,4-D was the optimal media for callus induction, which is on par with our studies on callus induction in petals. Datta et al., (2006) achieved high callus response in Taxus wallichiana on half WPM supplemented with 1.0–2.0 mg/L 2,4-D which was in accordance to our results for both leaf and petal explants.

A range of 2,4-D concentrations (0.1–2.0 mg/L) were used for callus induction from leaf and nodal segments of Cardiospermum halicacabum L. Even though high concentrations of 2,4-D (1.5 mg/L) was necessary for callus induction from leaf and nodal cuttings, it adversely affected further growth of the callus (Thomas and Maseena, 2006).

Table 1. Effect of 2,4-D Concentration on Callus Induction

Treatment	Concentration of 2,4-D mg/L	Callus induction Percentage ± SE
Treatment	Concentration of 2,4-D mg/L	Leaf	Petals
T1	0.5	20 ± 0.12	24 ± 0.14
T2	1.0	72 ± 0.14	48 ± 0.17
T3	1.5	100 ± 0.26	80 ± 0.22
T4	2.0	81 ± 0.13	100 ± 0.26
SEd		0.19	0.36
CD (0.05)		0.39	0.72

Table 2. Effect of 2,4-D Concentration on Callus Proliferation

Treatment	Concentration of 2,4-D mg/L	Callus Proliferation in mg ± SE
Treatment	Concentration of 2,4-D mg/L	Leaf	Petals
T1	0.5	21 ± 0.01	18 ± 0.09
T2	1.0	30 ± 0.08	27 ± 0.08
T3	1.5	40 ± 0.11	35 ± 0.09
T4	2.0	26 ± 0.09	43 ± 0.12
SEd		0.09	0.09
CD (0.05)		0.18	0.18

Induction of Somatic Embryos through Suspension Cultures

The friable green callus maintained in 1.0 mg/L BAP were subjected to suspension culture in liquid (without agar) MS medium supplemented with 1.0 mg/L BAP (Figure 3). This was subcultured every 2 weeks in the same medium. Somatic embryos were induced in all the calli after 2 weeks of inoculation in suspension culture. Cells in suspension show a faster multiplication rate than do cells in callus culture (Philips et al 1995). Gurel et al., (2002) obtained somatic embryos of sugar beet in suspension culture in medium supplemented with 0.25 mg/L BAP and 0.25 mg/L 2,4-D and concluded that increased concentrations of BAP increases the rate of cell division. Kumar et al., (2005), reported MS medium supplemented with BAP in suspension cultures to be favorable for callus and bud growth in Chrysanthemum.

Figure.3 Callus in Suspension Culture

Differentiation of Somatic Embryos and Regeneration

The regenerated somatic embryos differentiating in to globular stage, torpedo stage and cotyledonary stage were observed at various level of sub culturing.

The somatic embryos obtained from the suspension culture passed through cotyledonary stages. Tanaka et al., (2000) observed early torpedo, late torpedo and cotyledonary stages in somatic embryogenesis of D.grandiflora. The regeneration was observed to be 100%. The somatic embryos in the torpedo stage were transferred to MS basal medium for regeneration and cotyledonary stage embryos were obtained.

The embryos in the cotyledonnary stage were then transferred to MS medium supplemented with varying concentration of BAP. The results were recorded after 3 weeks and summarized in table 3.

The largest number of shoots (46.3 ±0.44) were found in MS medium supplemented with 2.0 mg/L BAP (T4) with significantly high plantlet formation compared to other concentrations. In MS media supplemented with 1.5 mg/L BAP (T3), 27.3 ± 0.36 shoots were observed while with 1.0 mg/L BAP (T2), 16.4±0.37 shoots were observed. The lowest number shoots of 9.4±0.30 was observed in 0.5 mg/L BAP (T1).

Table 3. Variation of BAP Concentration for Regeneration

S. No	Media + Concentration of BAP mg/L	Percentage of Number of shoots obtained ± S.E
T0	MS Basal	0.5±0.23
T1	MS + 0.5 BAP	9.4±0.30
T2	MS + 1.0 BAP	16.4±0.37
T3	MS + 1.5 BAP	27.3 ± 0.36
T4	MS + 2.0 BAP	46.3 ±0.44
SEd		0.53
CD		1.07

The effect of BAP was studied on the in vitro generated Chrysanthemum plantlets by Bhattacharya et al., (1999). He reported that the BAP in low concentrations gave good shoot induction in Chrysanthemum. Alizadeh et al., (2004) reported that MS media supplemented with 2.0 mg/L BAP, as the optimal media for shoot proliferation on embryo explants of wheat, which is comparable to Chrysanthemum regeneration in the present study.

Elongation and Rooting:

The somatic embryo regenerated plants were transfer in to elongation medium. The effects of KIN along with BAP were studied for effective elongation of shoot and root and the results were recorded after 1 month. For elongation MS medium supplemented with 0.1 mg/L BAP + 2.0 mg/L KIN was used and the elongation was maximum (19.66 ± 0.34). The effect of BAP and KIN were studied on the in vitro generated Chrysanthemum plantlets by Bhattacharya et al., (1999) and reported that the combination of BAP and IAA in low concentrations gave good shoot elongation in Chrysanthemum.After elongation the plants were transferred in to MS Basal medium (MS0). After 3 weeks profuse rooting was observed in all plantlets (Figure 4).

Figure.4 Rooting Plant

Hardening:

The rooted micro shoots were carefully removed and transferred to presoaked vermiculite for hardening initially inside a rectangular glass box, maintained for 3 days in the culture room and then transferred to the mist chamber for further establishment. Castillo et al., (2000) used peat-moss vermiculite for hardening and obtained satisfactory acclimatization. Good establishment of plants were seen after one week in the mist chamber.

CONCLUSION:

In conclusion, further investigations are necessary enhance the germination of somatic embryos ad subsequent establishment in the green house. This protocol will help in rapid propagation of Chrysanthemum for pharmaceutical product preparation. In vitro plant gave high amount of secondary metabolites in short period, so this may useful for the large scale production for pharmaceutical product.

REFERENCES:

1. Alizadeh H, Naghavi MR, Omidi M and Saatian B. Effect of plant growth regulators on direct shoot regenration of wheat (Triticum aestivum). Crop science society of America. Medison. 44; 2004:1839-1846.

2. Bhattacharya S, Das B, Ghose TK and Bhattacharya S. Investigation on seed germination of Nyctanthis arbortristis (Oleaceae) in relation to the total phenol content. Seed Sci. Technol., 27(1); 1999: 321-327.

3. Castillo P, Marquez J, Rubluo A, Hernandez G and Lara M. Plant regeneration from callus and suspension cultures of Valeriana edulis pp procera via simultaneous organogenesis and somatic embryogenesis. Plant science. 151; 2000:115-119.

4. Chen K, Plumb WG, Bennett NR and Bao Y. Antioxidant Activitives of Extracts from Five Anti-Viral Medicinal Plants. J. Ethnopharmacology. 96; 2005:201-205.

5. Datta MM, Majumder A and Jha S. Organogenesis and plant regeneration in Taxus wallichiana (Zucc.). Plant Cell Rep., 25; 2006: 11-18.

6. Gurel S, Gurel E and Kaya Z. Establishment of cell suspension cultures and Plant regeneration in sugar beet (Beta vulgaris L.). Turk J. Bot. 26; 2002:197-205.

7. Hitmi A, Coudret A and Barthomeuf C. The Production Pyrethrin by Plant Cell and Tissue Cultures of Chrysanthemum Cinerariaefolium and Tagetes species. Critical Reviews in Biochem. Mol. Biol. 35; 2000: 317-337.

8. Kumar A., Singh SP and Bhakuni RS. Secondary metabolites of Chrysanthemum genus and their biological activities, Current Science. 89; 2005: 1489-1501.

9. Murashige T and Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Planta. 15; 1962:472-497.

10. Obukosia SD, Kimani E, Wathaika K, Mutitu E and Kimani PM. Effect of growth regulators and genotypes on pyrethrum in vitro. In vitro cell Dev. Biol- Plant.11; 2004: 162-166.

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12. Tanaka K, Kudo S, KannoY and.Suzuki M. Somatic embryogenesis and plant regeneration in Chrysanthemum (Dendranthema grandiflorum (Ramat) Kitmura). Cell Biol. Morphogenesis. 19; 2000: 946-953.

13. Thomas TD and Maseena EA. Callus induction and plant regeneration in Cardiospermum halicacabum (L.) an important medicinal plant. Scientia Horticulturae. 108; 2006: 332–336.

Received on 05.01.2011 Accepted on 01.02.2011

Asian J. Pharm. Tech. 1(1): Jan.-Mar. 2011; Page 13-16

MATERIALS AND METHODS:

Callus induction and regeneration from Callus:

Somatic Embryogenesis in Suspension Culture:

Shoot Induction, Elongations and Rooting:

Hardening:

RESULT AND DISCUSSION:

Callus Culture:

Effect of 2,4-D Concentration on Callus Induction and Proliferation:

Table 1. Effect of 2,4-D Concentration on Callus Induction

Petals

Table 2. Effect of 2,4-D Concentration on Callus Proliferation

Hardening:

REFERENCES: