Tissue culture unite, Genetic Resources Department, Desert research Center (DRC)Cairo,. Egypt. ABSTRACT. An efficient method for dirat plant regeneration of Thymus capitatus L. was ..... informative book on herbs from around the globe.
Pak. J. Biotechnol. Vol. 5 (1-2) 39-44 (2008)
ISSN. 1812-1837
IN VITRO CLONAL PROPAGATION OF THYMUS CAPITATUS L. THROUGH DIRECT REGENERATION M. El-MAKAWY, M.YASSER, M.ABD ALLAH and S. NISHAWY Tissue culture unite, Genetic Resources Department, Desert research Center (DRC)Cairo, Egypt. ABSTRACT An efficient method for dirat plant regeneration of Thymus capitatus L. was developed by using shoot tips and stem node sections. Explants were cultured on MS basal medium fortified with various concentrations of cytokinins in single or combination forms. The best response for multiple shoot regeneration (6.28) was achieved on MS supplemented with 1.0 mgl-1 BA and 0.01 mgl-1 Zeatin after six weeks of shoot tips culture. While the best response for multiple shoots differentiated (12.4) within 8 weeks when stem node sections were cultured on MS medium containing 0.5 mgl-1 BA. High rate of root percentage (100%) was obtained in shoot regeneration cultures on ½ MS basal medium containing 3 mgl-1 IBA. Micropropagated plantlets were transplanted into soil for acclimation under greenhouse conditions. INTRODUCTION Thymus capitatus L. is an important species according to medicinal point of view. It contains high amounts of carvacrol or thymol or almost equal amounts of both phenols, depending on the habitat of the plant. The natural habitat unit is introduced as a tool for the assessment of the essential oil variation Regina et al., (2005). In medicinal uses, the leaves and essential oil are strongly antiseptic, deodorant and disinfectant (Huxley, 1992; Bown, 1995). The plant can be used fresh at any time of the year, or it can be harvested as it comes into flower and either be distilled for the oil or dried for later use. The essential oil should not be used in aromatherapy because it is highly irritant to the mucous membranes (Bown 1995). The essential oil, known as 'Spanish oregano oil', obtained from the leaves is also used in perfumery soaps, flavouring baked goods, condiments, beverages, ice creams and mouth wash (Uphof, 1959, Polunin, 1987, 1969, Huxley, 1992, Bown 1995, Facciola, 1990).
The plant is sometimes used as a condiment (Facciola 1990). Raw leaves are used in salads or added as a flavor to cooked foods. An aromatic tea is made from the leaves and the leaves are dried when the plants should be harvested in early and late summer just before the flowers open and the leaves should be dried quickly (Huxley 1992). The In vitro propagation technique has become one of the most ways reproducing crops that are difficult to propagate by conventional method such as seeds or cuttings. Micropropagation allows the production of large number of plants in a relatively small growing area and in a relatively shorter time. Micropropagation also produces rapidly and large quantities of the plants identical to their parents. The speed of tissue culture technology development has been accelerated by its particle commercialization for many crops (Nizar, 2001). Girija et al., (2006) used MS basal medium supplemented with BAP and Kintin as
40
Makawy et. al.,
cytokinin individually or in combination for direct multiple shoots of Ocimum sanctum L. differentiated within 6weeks. Arafeh et al., (2003) reported that the highest shoot proliferation of organium syriacum was obtained when Kin and BA were used. Soniya and Das (2002). reported that multiple of shoots Piper longum were induced from shoot tips cultured on agarbased Murashige and Skoog (MS) medium containing 4.44-22.19 µM benzyladenine (BA) and 4.64-13.9 µ M kinetin (K). Maximum number of shoots were induced with 8.9 µM BA and 4.64 µ M Kinetin were used as cytokinin in basal medium for further multiple shoots of Lepladenia retienlate (shekhawat et al., 2006). KiranGhanti et al., (2004) reported that A high frequency and rapid regeneration protocol was developed nodal explants of Mentha piperita on MS basal medium supplemented with BA 1 mgl-1. It was observed that the highest number of shoots was obtained on medium containing BAP. Khadeeva et al., (1995) reported that the average efficiency of micropropagation by nodal segments of Stachys sieboldii Mig could be increased to 1:100-1:200 on MS basal medium supplemented with 1 mg kinetin and 1 mgl-1 BA as a cytokinin, through the stimulation of adventitious shoot production. Oliphant, (1989); Pattanaik and chand (1996); Ramamurthy and Savithramma (2002); Usha-Mukundan et al., (2002); Phatak and Heble (2002); Nahida-chishti et al., (2006); Hembrom et al., (2006) and Rani et al., (2006) reported that shoots of Lavandula angustifolia, Ocimum americanum, Cassia alata,( Tylophora asthmatica and Uraria picta), Mentha aravensis, Lavandula officinalis, Pogostemon heaneanus Benth and coleus blumei Benth.were rooted best in half strength MS basal medium fortified with
Pak. J. Biotechnol
various concentration of IBA. Mohammadand Mohd (2005) reported that the regenerated shoots of Psoralea corylifolia rooted best on MS medium containing 0.5µ M IBA. In this study, a significant method of regeneration has been develop from shoot tipsand stem node of thymus capitatus L. MATERIAL AND METHODS Thymus capitatus L. Seeds were collected by Egyptian Desert Gene Bank (EDGB) from Saint Katharine region, South Sinai. Seeds were treated with sulphoric acid for 30 min then transferred to petri dishes for germination. After 10 days, seedling was developed and transferred into soil pots in greenhouse. Shoot tips (0.5-1cm) of about one years old plant were used as an ex-plant. Shoot tips were washed with running tap water for 1-2 hour then with sterilized distilled water contains few drops of liquid detergent in a flask or beaker and shack on magnetic stirrer for 15 min, then rinsed in sterilized distilled water to remove the soap. The explants were transferred to the laminar air flow hood and soaked in 5.25% of commercial sodium hypochlorite (20% NaOCl) for 20 min. After that washed with sterile distilled water to remove traces of the desinfection. Under aseptic conditions, explants (shoot tips and stem node sections) were cultured in Murashige and Skooge (1962 ) basal medium containing 3%(W/V) sucrose supplemented with different concentrations independently or in combination of BA (0.00,0.5 and 1.00 mgl1 ) and zeatin (0.00,0.01 and 0.02) mgl-1 .for establishment and multiplication. For rooting formation shoots were tested on root induction in Murashige and skooge (1962) salts media with addition of vitamins and 100 mg l-¹ myo-inositol, 30g l -¹ sucrose supplemented with
Number mean Of axillary shoot/ explant mean length of axillary shoot (cm)
0.00
0.00
0.77d
0.33c
1.52c
0.00
0.01
2.59c
1.23b
1.87c
0.00 0.5 0.5 0.5
0.02 0.00 0.01 0.02
d
1.66 4.88b 5.36ab 5.54ab
b
1.45 1.66b 2.29a 2.19a
1.96c 2.47b 3.29a 3.14a
1.00
0.00
4.92b
1.23b
1.9c
0.01 0.02
a
a
1.00 1.00
6.28 5.89a
2.42 2.72a
length of mean shoot (cm)
RESULTS AND DISSCUTIONS: Preliminary studies proved that Shoot tip explant were achived a high survival percentage 100% when exposed to 1.05% sodium hypochlorite (containing 5.25 % NaOCl ) for 20 min. Establishment and multiplication of thymus capitatus: Results presented in Table -1 showed the number of axillary shoot, length of axillary shoot and shoot length. Axillary shoot number, axillary shoot length and shoot length occure at a low frequency on MS basal medium without growth regulators . Although it was possible to induce and maintain shoots upon all media tested, the regeneration of axillary shoots (6.28), and shoot length (3.69) was optimal upon MS basal medium fortified with 1.00 mgl-1 BA and 0.01mgl-1 Zeatin length of shoot. The highest length of axillary shoot per explant (2.72cm) was achieved on MS basal medium supplemented with1.00 mgl-1 BA and 0.02 mgl-1 Zeatin . Girija et al., (2006) used MS basal medium supplemented with BAP and Kinetin as cytokinin individually or in combination for direct multiple shoots of Ocimum sanctum L. differentiated within 6weeks. The same result was obtained by Arafeh et al., (2003) and it was reported that the highest shoot proliferation of organium syriacum was obtained when Kin and BA were used. Soniya and Das (2002) reported that multiple of shoots Piper longum were induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.44-22.19 µM benzyladenine (BA) and 4.64-13.9 µ M kinetin (K). Maximum number of shoots
were induced with 8.9 µM BA and 4.64 µ M Kinetin. Table-1: Effect of various concentration independently or in combination of benzyl adenine and zeatin in different concentrations on establishment and multiplication stage of Thymus capitatus L. plant. Zeatin mgl-1
0.00,0.5,1.0,2.0 and 3.0 mgl-1 IBA. For Acclimatization the regenerated Thymus capitatus plants were transferred to the greenhouse during 2007 – 2008 according to ( Khalil, 2002).
In-Vitro clonal propagation of Capitatus L. 41
BA mgl-1
Vol. 5. (1-2) 2008
3.69a 3.18a
Stem node sections: Data in Table -2 showed that there is a high significant between treatment in all parameters. Nodes cultured on MS salts basal medium supplemented with 0.5 mgl-1 BA gave the best number of axillary shoot (12.4). In other hand the high length of axillary shoot per explant (3.24cm), length of shoot (4.58cm) were achieved from explants cultured on MS salts basal medium supplemented with1.00 mgl-1 BA and 0.01 Zeatin. While explants cultured on MS basal medium without growth regulators gave the least value in length of axillary shoot (1.14) was achieved on MS basal medium 0.01mgl-1 NAA ,while the least value of shoot length (1.14) was obtained on MS basal medium without growth regulators , but the least number of axillary shoot (3.33) was achieved on MS salts basal medium supplemented with 1.0 mgl-1 BA and 0.02 mgl-1 zeatin. The same results was obtained by shekhawat et al., (2006)
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Makawy et. al.,
Pak. J. Biotechnol
used BA and Kinetin as a cytokinin on MS basal medium for further multiple shoots of Leptadenia reticulate but results are similar to Kiran-Ghanti et al., (2004) and it was reported that A high frequency and rapid regeneration protocol was developed nodal explants of Mentha piperita on MS basal medium supplemented with BA 1 mgl-1. They found that The highest number of shoots (49.8) was obtained on medium containing BAP. Khadeeva et al., (1995) reported that The average efficiency of micropropagation by nodal segments of Stachys sieboldii Mig could be increased to 1:100-1:200 on MS basal medium supplemented with 1 mg kinetin and 1 mgl1 BA as a cytokinin, through the stimulation of adventitious shoot production.
3.43d 5.83c 4.54cd 12.4a 6.12c 5.47cd 8.92b 4.4cd 3.33c
1.14de 0.97e 1.26de 1.74c 2.47b 2.18b 1.46cd 3.24a 2.39b
length of mean shoot (cm)
0.00 0.01 0.02 0.00 0.01 0.02 0.00 0.01 0.02
mean length of axillary shoot (cm)
Zeatin mgl-1
0.00 0.00 0.00 0.5 0.5 0.5 1.0 1.0 1.0
mean Number Of axillary shoot/ explant
BA mgl-1
Table-2: Effect of interaction combination of benzyl adenine and zeatin in different concentrations on establishment and multiplication stages of Thymus capitatus L. plant.
1.31f 1.38f 1.49f 2.54d 3.3c 3.47bc 2.13e 4.58a 3.66b
Rooting of Thymus capitatus: Data in Table-3 revealed that there is a high significant in rooting percentage, root number and root length, whereas that half strength MS medium supplemented with 3.0 mgl-1 IBA produced the highest rooting percentage (100%). While the Optimum root length (1.87) was achieved on half
strength MS medium containing 0.5 mgl-1 IBA. The highest root number was achieved on half strength MS medium containing 2.0 mgl-1 IBA. Regenerated shoots cultured on MS basal medium failed to produce root system. This results were agreed whith that of Oliphant, (1989); Pattanaik and chand (1996); Ramamurthy and Savithramma (2002); Usha-Mukundan et al., (2002); Phatak and Heble (2002); Nahida-chishti et al., (2006); Hembrom et al., (2006) and Rani et al., (2006). They have reported that shoots of Lavandula angustifolia, Ocimum americanum, Cassia alata,(Tylophora asthmatica and Uraria picta), Mentha aravensis, Lavandula officinalis, Pogostemon heaneanus Benth and coleus blumei Benth were rooted best in half strength MS basal medium fortified (supplemented) with various concentration of IBA. But the least value in all parameters was achievd on MS basal medium supplemented with 0.5 mgl-1 IBA. in the contrary Mohammad-Anis and Mohd-Faisal (2005). reported that the regenerated shoots of Psoralea corylifolia rooted best on MS medium containing 0.5 µ M IBA
In-Vitro clonal propagation of Capitatus L. 43
Vol. 5. (1-2) 2008
Root number
Root length
0.34e 0.00e 2.44cde 5.45abc
0.32b 0.00b 0.3b 1.62a
4.33bcd
0.89ab
44.44cd
1.22de
1.21ab
ab
cde
1.87a 1.86a 1.15ab 0.88ab
IBA mgl-1
22.22de 0.00e 44.44 cd 66.67
Strength of MS
Root percentage
Table-3: Effect of interaction combination between strength of MS and IBA on rooting of Thymus capitatus L. plant
MS MS MS MS
0.00 0.5 1.0 2.0
MS
3.0
55.56bc
abc
d
Halfe MS Halfe MS Halfe MS Halfe MS Halfe MS
0.00 0.5 1.0 2.0 3.0
88.89 88.89ab 77.78abc 100 a
3.11 7.11ab 8.56a 8.11ab
Acclimatization: For acclimatization, plantlet were removed from rooting medium after six weeks of incubation and transferred to glass tube containing 10ml of water and maintained for a week in a naturally – illuminated acclimatization room at 25°C before transferring to the green house. The plantlets were soaked in a fungicide solution (1gl-1 of rizolex) for 3-5 min. The plantlets were then cultured in plastic pots 15 cm in diameter containing mixture of peat moss and sand 1:1 (V/V) and each pot was enclosed within a transparent plastic bag. After ten days the plastic bag was gradually perforated and eventually removed within one week. After 4 weeks the plants were transferred into plastic pots (30cm in diameter) and received appropriate nitrogen fertilizer. The plantlets were successfully acclimatized on peat moss and sand 1:1 (V/V) medium with 80% survival percentage of plants. This observation is in agreement with Reynold (1985) who recommended soaking of tissue culture derived plants in water prior to transplantation to the soil.
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mediated co-transformation of sugarcane. Arab. J. Biotech. 1: 19-23. Kiran- Ghanti, C.P. Kaviraj, R. B. Venugopal, F.T.Z. Jabeen and S. Rao. Rapid regeneration of Mentha piperita L. from shoot tip and nodal explants. Indian J.of Biotechnology. 3(4): 594-598. (2004) Mohammad, A. and F. Mohd. In vitro regeneration and mass multiplication of Psoralea corylifolia - an endangered medicinal plant . Indian J. of Biotechnology, 4(2): 261-264. (2005). Nahida, C. Z.A. Kaloo, A.S. Shawl and P.Sultan. Rapid in vitro clonal propagation of Lavendula officinalis chaix A multipurpose plant of industrial importance. Pakistan J. of Biological, Sciences. 9(3): 514-518. (2006) Nizar, A., Using plant tissue culture technique for rapid propagation . M.Sc. thesis, Fac. Agric., Cairo Univ. (2001) Oliphant, J.L. Micropropagation of Lavandula species. Combined Proceedings, International Plant Propagators Society, 37: 158-106 (1987). Pattnaik, S.K. and P.K. Chand. In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil).Plant Cell Reports 15(11): 846-850. (1996) Phatak, S. V. and M. R. Heble. Organogenesis and terpenoid synthesis in Mentha arvensis. Fitoterapia, 73(1): 32-39 (2002) Polunin. O. and Huxley, A. Flowers of the Mediterranean. Hogarth Press ISBN 0-70120784-1A very readable pocket flora that is well illustrated. Gives some information on plant uses. (1987) Polunin. O., Flowers of Europe - A Field Guide. Oxford University Press ISBN 0192176218An excellent and well illustrated pocket guide for those with very large pockets. Also gives some details on plant uses. (1969) Ramamurthy, N., and N.Savithramma, In vitro regeneration of a medicinal plant Cassia alata L. through axillary bud Journal of Plant Biology. 29(2): 215-218 . (2002) Rani, G., D. Talwar, A. Nagpal and G.S. Virk, Micropropagation of Coleus blumei from
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nodal segments and shoot tips. Biologia Plantarum. 50(4): 496-500. (2006) Regina, K., N.K. Dimitrios and K. Stella, Essential oil composition is related to the natural habitats: Coridothymus capitatus and Satureja thymbra in NATURA 2000 sites of Crete. International J. of Sience Direct. 66 (22): 2668-2673. Reynold, J. P., Vegetative propagation of palm trees, In: Bonga, J.M. and Duzan D.J. (eds), Tissue culture in Forestery, Nithoff/Junk the Hage, PP. 182-207. (1985). Shekhawat, N.S., A. Kackar, M.S. Rathore, M. Singh, H.R. Dagla and Vinod-Arua, Establishment and economic evalution of micropropagated jeewanti (Leptadenia reticulate wight & Arn.) plants in field. Natural Product Radiance.5(4): 311314.(2006) Soniya, E.V. and M.R. Das, In vitro micropropagation of Piper longum - an important medicinal plant. Plant Cell Tissuen and Organ Culture. 70(3): 325-325-327 (2002). Uphof. J. C., Th. Dictionary of Economic Plants. An excellent and very comprehensive guide but it only gives very short descriptions of the uses without any details of how to utilize the plants. Not for the casual reader. (1959) Usha, M. L. Sivaram and A. Kumar, Micropropagation of Tylophora asthmatica and Uraria picta. Plant Cell Biotechnology and Molecular Biology 3(1/2): 73-76. (2002)
