Thidiazuron Induced Direct Regeneration from Leaf Explants of Scoparia dulcis L. A Pharmaceutical Plant

 

Karthikeyan Subbarayan1, Rajagopal Kalyanaraman1, Prathima Badrinarayanan1, Rajasekaran Murugan2, Gowri Karthik1 and Gomathee Ravichandran1

1Department of Biotechnology, School of Life Sciences, VELS University, Chennai-600117, India 2Group of Soil Biology and Plant Nutrition, Faculty of Organic Agricultural Sciences, University of Kassel, Nordbhanhof str 1a, D-37213 Witzenhausen, Germany

*Corresponding Author E-mail: karthisubbarayan@gmail.com

 

ABSTRACT:

Leaf explants of Scoparia dulcis L. gave rise to multiple shoots when cultured on MS medium supplemented with different concentrations of TDZ and IAA. The highest rate of shoot multiplication was obtained in MS medium containing 4.0 M TDZ and 1.0 M IAA (26.6 0.98). Differentiated shoot buds elongated to 5.8 cm in 21 days in 9 M KIN amended medium. The regenerated shoots were rooted on half-strength MS basal medium with different concentrations of IBA and IAA. The maximum number of roots was achieved on the medium containing 2.8 M IBA. In vitro regenerated plantlets were transferred to plastic pots containing coco peat as a potting mix and were thereafter successfully established under ex vitro conditions. The survival percentage of transplanted plantlets was 90.6%.

 

KEYWORDS: Scoparia dulcis, medicinal plant, leaf explants, micropropagation, Thidiazuron.

 


INTRODUCTION:

Medicinal plants are of great interest to the researchers in the field of biotechnology as most of the drug industries depend, in part, on plants for the production of pharmaceutical compounds1. Among the world's 25 best selling pharmaceutical medicines, 12 are plant derived2,3. Scoparia dulcis L. commonly known as ' Sarkarai Vembu in Tamil and sweet broom in English (Family: Scrophulariaceae) a small erect, slender, rigid, perennial herb with three serrate-margined, ovate-elliptic leaves at each node, small white, axillary, solitary flowers and small coriander-like fruits4,5. Traditionally Scoparia dulcis has been used as a remedy for treating stomach ailments, hypertension, diabetes, inflammation, bronchitis, hemorrhoids, hepatosis, an analgesic and antipyretic agent6,7. Hot water infusion and decoction of the leaves or whole plant is used medicinally by indigenous tribes of Nicaragua to treat malaria, stomach disorders, menstrual disorders, insect bites, fevers, heart problems, liver disorders, and venereal disease also for blood cleansing8. Moreover the plant is reported in Siddha system of medicine to treat diabetes and to treat any poisonous bite9.

 

Mucilage is released when the whole plant is soaked in water, thus helping to protect and regenerate normal cells; it may also act as an immunostimulator. The plant has been used to treat skin rashes in Martinique and Trinidad for irritated skin in Brazil, as a multi-ingredient preparation for treating burns in eastern Nicaragua and to kill lice and fleas and used against vermin in Paraguay10. The dried roots and aerial parts of Scoparia dulcis contain hydroxamic acids which provide insect, fungal, and bacterial resistance11. The scientific research reveals numerous chemical studies on isolated chemicals include coumarins, phenols, saponins, tannins, amino acids, flavonoids, terpenoids and catecholamines7. The terpenoids are responsible for numerous medicinal effects. Scoparic acid A, Scoparic acid B, Scopadulcic acid A, Scopadulcic acid B, Scopadulciol and Scopadulin are all biologically active. These chemical compounds have various biological activities, including inhibition of the replication of herpes simplex virus (The mechanism of action is unknown but does not involve a direct virucidal effect or inhibition of virus attachment), inhibition of proton pumps, potassium adenosine triphosphate (ATP)ase activator and antitumor promoting activity. The acetylated flavone glycosides from broomweed have nerve growth factor (NGF)-potentiating activity or neurotrophic activity that may be useful in treating neurological disorders. The flavone glycosides including isovitexin also inhibit β-glucuronidase12. The plant has been proved for its antidiabetic and antioxidant activity13.

The over exploitation of medicinal plant leads to habitat loss, extinction and reduces species size. Besides that they are prone to environmental catastrophe, demographic or loss of genetic variations and accumulations of deleterious mutations14. Mass propagation of plant species through in vitro culture is one of the best and most successful examples of commercial application of plant tissue culture technology. Recently, there has been much progress in this technology for some pharmaceutically important medicinal plants. Thidiazuron (TDZ), a urea-derived cytokinin, is a potent cytokinin for plant tissue culture15 and is extensively used for the induction of shoot regeneration in several plant species16,17,18. While micropropagation through auxiliary shoot proliferation3,5,19 of Scoparia dulcis has been reported, the study using other explant sources have not been reported. Hence, the present investigation reveals the invitro regeneration from leaf explant of Scoparia dulcis.

 

MATERIALS AND METHODS:

Plant materials:

Scoparia dulcis was freshly harvested from Madras Christian College campus, Chennai, India and their leaves were used as explant source. The explants were washed with soap (soap powder) in running tap water for 1 hour. This is necessary to remove the exudates present within the tissues. The explants washed with Tween 20 (2%, v/v) and rinsed until traces of soap was removed. Then the explants were transferred to a sterile laminar flow and surface sterilized the explants using mercuric chloride (0.1%, w/v) followed by three washes with sterile distilled water. The leaves were trimmed into pieces of about 0.5 cm2 and inoculated on to culture medium.

 

Culture medium and conditions:

Murashige and Skoog medium (1962)20 basal medium was supplemented with various plant growth regulators, 3.0% sucrose and 0.8% agar. The pH was adjusted to 5.8 and autoclaved at 121C under 15 lb pressure for 15-20 minutes. Explants were placed in culture tube and incubated at 242C and 60% relative humidity under the light 16 hour-day-1 photoperiod, provided light intensity of 2000 lux using a white fluorescent light.

 

Shoot Proliferation media:

MS medium supplemented with TDZ (1.0, 2.0, 3.0, 4.0 and 5.0 M) was used for microshoots formation from leaf explants and the cultures. Number of new shoot proliferation of each culture was recorded after every week of inoculation.

 

Root induction:

For in vitro rooting, individual shoots (3 - 6 cm) were excised from the proliferated shoot cultures and implanted onto half strength of MS with different concentrations and combinations of IBA (Indole butyric acid) and IAA (Indole acetic acid) (Table 1).

 

Acclimatization and hardening:

The formation of healthy shoots and roots make sure that this is ready to harden. The rooted plants were removed from the culture tubes, washed with sterile distilled water, and transferred to protrays with sterile cow dung: coca peat: sand (1:1:1 v/v/v). The plantlets were placed at 70 to 80% humidity, 25 2C under a 12-hours photoperiod for acclimatization.

 

For hardening, the rooted plants were removed from protrays, washed with sterile distilled water and transferred to green house. These hardened plants were transferred to the field and the survival rate was recorded19. Twenty cultures were used per treatment and each experiment was repeated at least three times. Percentage of success was scored four weeks after culture. The effects of different treatments were quantified and the data subjected to statistical analysis.

 

RESULT:

Within seven to 15 days of culture microshoots were formed from leaf explant on MS supplemented with 2 5 M TDZ either alone or in combination with 0.5 2 M IAA (Table 1). Maximum number of microshoots was 26.6 0.98 per culture developed on MS with 4 M TDZ + 1 M IAA after four weeks (Fig. l). Elongation of shoots was observed from the microshoots when subcultured on MS + (6-12) M KIN after four weeks (Table 1). The highest length of shoot was 5.8 0.78 developed on MS with 9 M KIN (Table 2, Fig. 2). The mean values of root induction from shoots of S. dulcis cultured in half strength MS medium with different concentrations of IBA and IAA are given in Table 3. In the case of IAA, maximum root induction was noticed at a concentration of 2.8 M (Fig. 3), whereas in half strength MS medium with IBA showed maximum root induction at a concentration of 2.4 M. Though root length was not much differ between IAA and IBA treatments, IBA produced better results than IAA. During the process acclimatization and hardening, about 85 % survival in chamber culture and about 90% survival in both greenhouse (Fig. 4) and field were noticed. The regenerated plants were phenotypically normal. 20 explants and culture were maintained in each treatment and data (SE) were recorded up to four weeks of culture

 

Fig. 1 Fig. 2

Fig.1. Shoots formation started from leaf explants on MS medium with 4 M TDZ + 1 M IAA after four weeks

Fig. 2. Shoot elongation on MS with 9 M KIN after three weeks of subculture


Table 1. Effect of different concentrations and combinations of growth regulators on MS for microshoots from the leaf explants of S. dulcis.

 

Growth regulars (M) TDZ IAA

% of explants producing microshoots

Mean No. of shoots/culture (Mean SE)

2.0

22.6

5.7 0.83

3.0

44.8

11.3 0.75

4.0

86.2

20.6 0.96

5.0

66.6

12.2 0.62

4.0 0.5

86.8

23.7 0.86

4.0 1.0

88.6

26.6 0.98

4.0 1.5

64.2

14.2 0.73

4.0 2.0

38.4

6.7 0.74

 


 

Table 2. Effect of different concentrations KIN on MS for elongated shoots from the microshoots of S. dulcis.

 

Growth regular KIN (M)

% of microshoots producing elongated shoots

Average length (cm) of shoots (Mean SE)

6.0

36.8

2.7 0.92

7.0

48.4

4.3 0.77

8.0

86.4

4.6 0.86

9.0

94.2

5.8 0.78

10.0

76.6

4.2 0.32

11.0

54.8

3.3 0.77

12.0

32.0

2.4 0.61

Fig. 3 Fig. 4

Fig. 3. Rooting of in vitro regenerated shoots in half strength of MS with 2.8 M in four weeks

Fig. 4. Acclimatized regenerated plant of two months old in green house condition

 

Table 3. Root induction at different concentrations of auxin in half strength MS medium from shoots of S. Dulcis

 

Growth regulators (M)

% of rooting response

Mean No. of roots/shoot

Average length of roots (cm)

IAA

2.0

82.4

4.1 0.91

1.6 0.75

2.4

89.6

6.1 0.87

2.1 0.85

2.8

78.2

3.2 0.77

1.7 0.74

3.0

72.6

2.8 0.96

1.5 0.95

IBA

2.0

82.8

3.8 0.82

1.8 0.81

2.4

90.6

6.4 0.74

2.4 0.72

2.8

82.8

4.7 0.97

1.6 0.87

3.0

79.6

2.6 0.68

1.3 0.93

 

DISCUSSION:

TDZ, a synthetic phenylurea, is considered one of the most active cytokinins for shoot induction in plant tissue culture15,21. TDZ-induced shoot regeneration from different explants of many recalcitrant species as well as from medicinal plants has been reported22-25,18,26,27, suggesting that TDZ results in shoot regeneration better than other cytokinins28,27. TDZ-induced morphogenesis probably depends on the levels of endogenous growth regulators, and TDZ modulates endogenous auxin levels29,30. Direct shoot multiplication is preferred for generating true-to-type plants compared with callus regeneration. The results also correlate with the KIN-induced regeneration of S. dulcis from nodal explants19. IBA is the most commonly used auxin for root induction; its superior role in this function has been reported in several plants31,32,27,30. This study reports a simple micropropagation protocol and the rapid in vitro multiplication from leaf segments of the useful medicinal plant- S. dulcis. Shoots can be easily derived from node cultures on TDZ containing medium and subsequently elongated with KIN and rooted on IBA containing medium. Both shoot and root can be derived from node cultures on KN and IAA containing medium. This investigation offers a means for producing more identical plantlets from leaf explants of S. dulcis.

 

ACKNOWLEDGEMENT:

Authors are thankful to the Management of Vels Educational Trust, Chennai, Tamilnadu, India, for providing the infrastructure for the present study.

 

REFERENCES:

1.       Chand S, Sahrawat AK and Prakash DVSSR. In vitro culture of Pimpinella anisum L. (Anise) J. Pl. Biochem. Biotech. 1997. 6: 1-5.

2.       'O' Neill M and Lewis A. Human medicinal agents from plants. In : Kinghorn AD Balandrin MF, ACS Sysmposium Series 534, Washington, DC. 1993. pp. 48.

3.       Hassan AKM, Afroz F, Bari LS, Munshi JL, Jahan MAA and Khatun R. Callus Induction and High Frequency Regeneration of Plantlets of Scoparia dulcis L., a Perennial Medicinal Herb, Through Auxiliary Shoot Proliferation. Plant Tissue Culture and Biotechnology. 2008. 18(1): 75-83.

4.       Ghani A. Medicinal Plants of Bangladesh with Chemical Constituents and Uses. 2nd Ed. Asiatic military press, Dhaka. 1998.

5.       Hassan AKM, Bari LS, Sultana R, Begum N and Khatun R. In Vitro Clonal Propagation of Scoparia dulcis L., a Perennial Medicinal Herb. Bangladesh J. Sci. Ind. Res. 2009. 44 (3): 341-346.

6.       Riel MA, Kyle DE and Milhous WK. Efficacy of scopadulcic acid A against Plasmodium falciparum in vitro. J Nat Prod. 2002. 65: 614-615.

7.       Ratnasooriya WD , Jayakody JR , Premakumara GA and Ediriweera ER. Antioxidant activity of water extract of Scoparia dulcis. Fitoterapia. 2005. 76:220-222.

8.       Latha M, Pari L, Sitasawad S and Bhonde R. Insulin-secretagogue activity and cytoprotective role of the traditional antidiabetic plant Scoparia dulcis (Sweet Broomweed). Life Science. 2004. 75: 2003-2014.

9.       Mohan VR, Rajesh A, Athiperumalsami T and Sutha S. (2008). Ethnomedicinal Plants of the Tirunelveli District, Tamil Nadu, India. Ethnobotanical Leaflets. 2008. 1:1-18.

10.     Lans C, Harper T, Georges K and Bridgewater E. Medicinal plants used for dogs in Trinidad and Tobago. 2000. Prev Vet Med. 45: 201-220.

11.     Pratt K, Kumar P and Chilton WS. Cyclic hydroxamic acids in dicotyledonous plants. Biochem Syst Eco. 1995. 23:781-785.

12.     Li Y, Chen X, Satake M, Oshima Y and Ohizumi Y. Acetylated flavonoid glycosides potentiating NGF action from Scoparia dulcis. J Nat Prod. 2004. 67:725-727.

13.     Pari L, Latha M and Rao CA. Effect of Scoparia dulcis extract on insulin receptors in streptozotocin induced diabetic rats: studies on insulin binding to erythrocytes. J Basic Clin Physiol Pharmacol. 2004. 15:223-240.

14.     Frankham R. Inbreeding and extinction: a threshold effect. Conservation Biology. 1995. 9: 792 799

15.     Huetteman CA and Preece JE. Thidiazuron: a potentcytokinin for woody plant tissue culture. Plant Cell Tiss. Org. Cult. 1993. 33: 105-119.

16.     Li H, Murch SJ, and Saxena PK. Thidiazuron induced de novo shoot organogenesis on seedlings, etiolated hypocotyls and stem segments of Huang-qin. Plant Cell Tiss. Org. Cult. 2000. 62: 169-173.

17.     Mohan, M.L. and Krishnamurthy KV. Somatic embryogenesis and plant regeneration in pigeonpea. Biol. Plant. 2002. 45: 19-25.

18.     Liu CZ, Murch SJ, Demerdash EL and Saxena PK. Regeneration of the Egyptian medicinal plant Artemisia judaica L. Plant Cell Rep. 2003. 21: 525-530.

19.     Karthikeyan S, Prasad R, Mahendran TS, Rajagopal K and Ravendran V. Direct regeneration and in vitro flowering of Scoparia dulcis L. Indian J.Sci.Technol. 2009. 2(5): 55-57.

20.     Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum. 1962. 15: 473-497

21.     Murthy BNS, Murch SJ, and Saxena PK. Thidiazuron: a potential regulator of in vitro plant morphogenesis. In Vitro Cell. Dev. Biol. Plant. 1998. 34: 267-275.

22.     Bhagwat B, Vieria LGE, and Erickson LR. Stimulation of in vitro benzyladenine and gibberellic acid. Plant Cell Tiss. Org. Cult. 1996. 46: 1-7.

23.     Seneviratne P and Flagmann A. The effect of thidiazuron on axillary shoot proliferation of Hevea brasiliensis in vitro. J. Rubber Res. Inst. Srilanka. 1996. 77: 1-14.

24.     Pelah D, Kaushik RA, Mizrahi Y and Sitrit Y. Organogenesis in the vine cactus Selenicereus megalanthus using thidiazuron. Plant Cell Tiss. Org. Cult. 2002. 71: 81-84.

25.     Schween G and Schwenkel HG. In vitro regeneration in Primula ssp. via organogenesis. Plant Cell Rep. 2002. 20: 1006-1010.

26.     Mithila J, Hall JC, Victor JMR and Saxena PK. Thidiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (Saintpaulia ionantha Wendl.). Plant Cell Rep. 2003. 21: 408 414.

27.     Thomas TD. Thidiazuron induced multiple shoot induction and plant regeneration from cotyledonay explants of mulberry. Biol. Plant. 2003. 46: 529-533.

28.     Barna KS and Wakhlu AK. Effect of thidiazuron on micropropagation of rose. In Vitro Cell. Dev. Biol. Plant. 1995. 31: 44-45.

29.     Hutchinson MJ and Saxena PK. Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium hortorum Bailey) tissue culture. Plant Cell Rep. 1996. 15: 512-515.

30.     Thomas TD and Puthur JT. Thidiazuron induced high frequency shoot organogenesis in callus from Kigelia pinnata L. Bot. Bull. Acad. Sin. 2004. 45: 307-313.

31.     Fracaro F and Echeverrigaray S. Micropropagation of Cunila galioides, a popular medicinal plant of south Brazil. Plant Cell Tiss. Org. Cult. 2001. 64: 1-4.

32.     Abrie AL and Staden JV. Micropropagation of the endangered Aloe polyphylla. Plant Growth Regul. 2001. 33: 19-23.

 

 

 

Received on 27.03.2010 Modified on 23.04.2010

Accepted on 29.05.2010 RJPT All right reserved

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1099-1102