View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Journal of Advanced Applied Scientific Research (JOAASR) Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28 In vitro rapid multiplication of Coldenia procumbens L. from shoot tip explants G. Jahirhussain*, S. Palanivel , V. Tamilselvan, V. Muniappan, K. Deepa, R. Veerappan P.G. and Research Department of Botany, Government Arts College (Autonomous), Karur -639 005. Tamilnadu, India. Corresponding Author E. mail: [email protected] Abstract: An efficient protocol for in vitro rapid multiplication of Coldenia procumbens L. has been developed. The shoot tip explants were cultured on MS basal medium supplemented with different concentrations of BAP and KIN. The two cytokinins are tested, BAP was found to develop higher number of shoots from the shoot tip explants when compared to KIN. Higher number of shoots was produced from all the concentrations of BAP and KIN. The highest frequency of 100% shoot induction was observed on MS basal medium supplemented with 6 μM BAP and 10 μM KIN. The number of shoots observed on the MS basal medium supplemented with BAP obtained 14 as well as 11 in KIN. The data's are recorded after 30days of inoculation. Key words : Coldenia procumbens L., cytokinins, shoot tip explants. 18 Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28 INTRODUCTION grounds, stems reaching 10-50cm long, Coldeniaprocumbens Linn.(Boraginaceae) shaggy with white hairs, branches often is an annual herb, common weed in India. numerous. It is distributed in tropical and It is found widely in south India on waste subtropical zones and found widely in south lands, common in dry rice grounds. The India3-6. Leaves: Crisped, alternate, short [1] [2] genus having 24 species of prostate. and sessaile 2-6×1.2-2cm, obovate to Coldenia procumbens is only species of its oblong, rounded at the apex, warty hair with genus has a place both in the Hortus rosette of basal cells, stomata anomocytic, Bengalensis and Moon’s Catalogue of palisade adaxial, veins 4-6 pairs on each [3] [5],[6] ceylon. This plant is widely used in side. Flowers: Small, 3-4mm long, traditional medicines in india, Africa, white, sessile, inconspicuous, solitary, malaysia. Acetone, water, methanolic axillary, nearly sessile; calyx- 1-1.5cm, extract of dried aerial parts shows weak lobes lanceolate to ovate lanceolate, corolla angiotensin-converting enzyme inhibition a pale yellow and very small.Sepals-5, in vitro. [4] broadly lanceolate, 2.5mm long, petals-5, Taxonomic classification of Coldenia united below into a short tube, stamens-5, procumbens Linn. inserted styles-2.[6],[7] Fruit:3-4mm long, Kingdom: Plantae Composed of four cells wrapped in calyx, Division: Tracheophyta with single seed in each cell. Fruit initially Class: Magnoliopsida splitting into 2 halves, later each half into 2 Order: Boraginales one seeded nutlets with distinct beak.[3] Family: Boraginaceae Fresh leaves of Coldenia Genus: Coldenia procumbens ground and applied to Species: Coldenia procumbens Linn. Rheumatic Swellings, equal parts of dried Synonyms: Creeping Coldenia; Ayurvedic powder mixed with seeds of fenugreek : Tripakshee; causes Suppurations of boils. Fresh leaves Vernacular Names: are used to promote maturation, the Sanskrit - Tripakshee ; decoction of Coldenia procumbens leaves English - Creeping Coldenia when mixed Hindi - tripungkee; with Centella asiatica and Madhuca Telugu -hamsapadu ; longifolia and Ixora coccinia would give Tamil - seruppadai or cherupadai significant result in wound healing, plant It is a prostrate herb usually lying extract also used in treatment of piles, [1] ,[2], [3], [4], [8] ,[9] quite flat on the ground,common on dry rice Leucorrhoea, Menorrhagia. 19 Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28 Coldenia procumbens having the Materials and Methods glycosides, phytosterols, proteins, amino Source of Explants acids, fixed oils flavonoids, gums and The field grown Coldenia mucilage as a chief constituents. Alkaloids procumbens Linn. (Boraginaceae) was and tannins are higher in alcoholic extract selected for the source of explants in the than in water extract. Reducing sugars and present Study. Shoot tip segments were phenols are higher in water extract than in used as explants for micropropagation. alcoholic extract. Non-reducing sugars and steroids are equally present in both the Culture Medium extracts. Saponins and fixed oils & fats are The nutrient medium consists of present only in water and alcoholic extracts inorganic salts, carbon source and organic [10] ,[11] respectively. supplements. In addition, vitamins and Micropropagation is one of the growth regulators are also added to the important tool for conservation of medium. In the present study, the basal medicinally important plants. It’s also a medium consists of the mineral salts and viable tool for rapid multiplication within a organic nutrients of Murashige and Skoog short duration. It is a method for production [14] [15] (MS) salts with B5 vitamins are used. of clones. This method conducted under For convenience, throughout this chapter, laboratory condition and it has a chance to MS medium with MS salts plus B5 vitamins produce genetically homogenous plants. is being referred as MS medium. The basal Micro propagation method is highly impact medium is supplemented with various in Plant breeding, Horticulture and concentrations and combinations of Forestry. It is a suitable method for different growth regulators. obtaining a large quantity of genetically homogenous and healthy plant material Growth regulators [12] which can be used for planting. This The prwsent study the hormone technique is an alternative method of concentration was used in µM (micromole). propagation as there is an increase in the These growth regulators were used as supplement to the basal medium propagation rate of plants, availability of individually as well as in different plants throughout the year, protection of combinations. plants against pests and pathogens under Cytokinins: BAP (6benzylaminopurine) controlled conditions and the availability of KIN (6-furfurylaminopurine) uniform clones and uniform production of [13] secondary metabolites. 20 Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28 Preparation of culture medium Sterilization of explant The present study was performed in the The explants were taken from the field basal medium with MS salts, B5 vitamins, grown mature plants. The explants 3% sucrose and 0.8% agar.The basal consisting of the shoot tip were surface medium was variously supplemented with sterilized by rinsing in running tap water for factorial combinations of different growth 30 minutes. Then they were washed in an regulators ranging from 2 – 10 µM BAP and agitated solution of liquid detergent KIN for shoot multiplication. After adding (Teepol) for 5 minutes and followed by all the supplements (various concentrations distilled water for 2-3 times for removing of different hormones) to the basal medium, the traces of liquid detergent. After the pH of the medium was adjusted to 5.6. thorough washing, the materials were taken Sterilization of Culture Medium and in to the Laminar Flow Chamber where they Glassware's were disinfected with 70% alcohol for 30- 60 seconds followed by 0.1% mercuric The culture medium containing high chloride for 3-5 minutes. Finally, the concentration of sucrose supports the materials were thoroughly rinsed with growth of several microorganisms. These sterile distilled water for 4-5 times to microbes generally grow much faster than remove the traces of mercuric chloride. the explants and finally spoil the culture. So it is very essential to maintain a complete Inoculation Procedure aseptic environment inside the culture tube. Before starting inoculation all the Therefore, the culture medium, glassware's, requirements such as culture tubes, forceps and scalpels was sterilized by containing media, spirit lamp, sterile water, autoclaving at 1.06 kg cm-2 and 121°C for glassware and explants, were placed in the 15 min. As well as the same procedure to laminar air flow chamber. The platform follow the sterilization of. During this surface of the chamber was swapped with period much care was taken to avoid 70% alcohol. After swapping the chamber denaturation of growth regulators and with 70% alcohol, the UV light was vitamins that were incorporated into the switched on for 30 minutes. After 30 medium. The culture tubes left free until minutes, the UV light was switched off and agar in the medium become solidified. Then the white fluorescent light was switched on. the tubes were transferred to inoculation Before inoculation, hands were rinsed with chamber for inoculation. absolute alcohol. The instruments were 21 Journal of Advanced Applied Scientific Research - ISSN: 2454-3225 G. Jahirhussain et.al JOAASR-RTB-2016- February-2016:18- 28 sterilized by dipping in absolute alcohol combinations or whichever is the best for followed by flaming and cooling. further multiplication. To facilitate higher number of shoot formation, the explants The inoculation was carried out in were also subcultured on conical flasks the vicinity of flame. The surface sterilized and/or culture bottles which