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Vitro Grown Microshoots and Callus Cultures of Ammi Visnaga (L.) Lam

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Vitro Grown Microshoots and Callus Cultures of Ammi Visnaga (L.) Lam Volume 12, Number 1,March 2019 ISSN 1995-6673 JJBS Pages 43 - 48 Jordan Journal of Biological Sciences Investigating the Antimicrobial Potential of in- vitro Grown Microshoots and Callus Cultures of Ammi visnaga (L.) Lam. Majd M. Al-Saleh1, Rida A. Shibli1*, Hamzah M. Al-Qadiri2, Reham W. Tahtamouni3, Maysaa M. Darwish4 and Tamara S. Al- Qudah1 1 Hamdi Mango Center for Scientific Research (HMCSR),2Department of Nutrition and Food Technology, School of Agriculture, University of Jordan, 3Department of Applied and Social Sciences, Princess Alia University College, Al-Balqa Applied University, 4National Center for Agriculture Research and Extension, Amman, Jordan.. Received May 9, 2018; Revised July 1, 2018; Accepted July 10, 2018 Abstract Ammi visnaga (L.) Lam is a valuable herbal plant that is frequently collected for medicinal purposes. This study is conducted to evaluate the antimicrobial potential of the in-vitro grown microshoots and callus cultures of this plant against selected strains of bacteria and fungi. Shoot multiplication was obtained in MS medium containing 0.5 mg/L BA + 0.1 mg/L NAA, while callus multiplication was performed on MS medium containing 1.0 mg/L BA + 2.0 mg/L 2,4-D under light conditions. The aqueous and methanolic extracts were prepared from both culture types in addition to in-vivo grown plant material to experiment their antimicrobial activities. Generally, the in-vitro extracts of the microshoots and the callus cultures acted best against bacteria compared to the field plant extract. Moreover, the methanolic extracts were generally found to exhibit far better results and resistance against the tested microbes than the aqueous extracts. C. albicans was the most sensitive species to the microshoots extracts followed by gram-positive bacteria at MIC values of (3.125, 6.25) mg/mL. Meanwhile, E. coli bacteria were most sensitive to the microshoot extracts as they were completely inhibited at MIC value of (0.78 mg/mL). Moreover, the results of the callus extracts showed that C. albicans was the most sensitive at MIC values of (1.56 and 3.125) mg/mL, followed by gram-positive bacteria. On the other hand, gram-negative bacteria were the most resistant microbes to all experimented extract types in this study. Keywords: Ammi visnaga , Antimicrobial activities, Callus, Microshoots Habitat destruction as a result of population growth, 1. Introduction urbanization and uncontrolled collection caused overexploitation of wild plants particularly medicinal Ammi visnaga (L.) Lam is a herbaceous medicinal plant plants (Al-Quran, 2011). Moreover, about 90 % of the which belongs to the family Apiaceae. It is found mainly natural resources used by industries are collected from in the Mediterranean regions, and is also distributed wild plants. This is due to the difference in price between abundantly throughout the world as an introduced species. the wild and cultivated plants, which could explain the A. visnaga (L.) Lam is used frequently in many countries lack of favorable plant materials, or to the high demands as a herbal medicine for different purposes. Ancient for the wild materials, which resulted, in turn, in the records revealed various medicinal properties of this plant destruction of medicinal plants. Therefore, it has become as a popular source to cure a variety of different ailments an urgent matter to take legal measures and establish a (Hashim, et al., 2014). compact program for the sake of saving all medicinal Phytochemical studies on Ammi visnaga revealed the plants (Craker, 2007; Sharma et al., 2010; Tahtamouni, et diversity of its chemical constituents, as they comprise al., 2015). several groups each with several compounds. The most The recent development in biotechnology particularly important one includes Khellin (0.3-1.2 %) from the in the techniques of plant tissue culture have come as a furanochromones group (2-4 %) (Hammouda et al., 2005; boon to cope up with this alarming situation and to WHO, 2007; Sellami et al., 2013; Hashim et al., 2014; overcome these problems through the rapid Talaat, et al., 2014). It is worth mentioning that, micropropagation method in order to save the natural furanochromones particularly visnagin, khellin, and herbal wealth (Sridhar and Aswath, 2014). khellol-glucoside are exclusively extracted from two Initially, plant tissue culture was exploited as a research species only, which are A. visnaga (L.) Lam tool and was focused on attempts to culture and study the (Umbelliferae) and Eranthis hyemalis L. (Ranunculaceae) development of small, isolated segments of plant tissues or (Kaul and Staba, 1967; El-Fiky et al., 1989). isolated cells. Plant tissue culture is now a well-established * Corresponding author. e-mail: [email protected]. 44 © 2019 Jordan Journal of Biological Sciences. All rights reserved - Volume 12, Number 1 technology. Like many other technologies, it has gone temperature with 200 rpm. Then the alcoholic extract was through different stages of evolution; scientific curiosity, filtered using vacuum buchner funnels, and was research tool, novel applications and mass exploitation concentrated in a rotary thin-film evaporator and stored in (Idowu et al., 2009). a refrigerator in a stand manner to precipitate for a few Although the discovery of antibiotics was a turning days until the formation of three layers: an upper oily layer point that revolutionized medicine in human history removed by filtration with a gentle suction and a middle (Davies and Davies, 2010), the uncontrolled use of cream, a fatty layer removed by the help of petroleum antibiotics in addition to the their side effects on human ether and a lower green crystalline layer from where pure health led to an imperative necessity to find therapeutic khellin was obtained. The extracted khellin was checked alternates such as semisynthetic antibiotics (Be´rdy, 2012), based on the fact that mixing 5-8 mg of khellin with a or plant-derived antimicrobials (Upadhyay et al., 2014). small piece of solid NaOH will produce a distinct rose-red More than a quarter of the modern medicines are derived color (Barakat, And Badran, 1951; Hassan and Zubair, from medicinal plants either directly or indirectly through 1980; Kar, 2003). the new technology applications of folk knowledge. This 2.2. In-Vitro Multiplication percentage can reach 60 % in the case of antitumoral and antimicrobial pharmaceuticals (WHO, 2011). 2.2.1. In-vitro Multiplication of Microshoot and Callus Several researches have been conducted recently for the Cultures extraction of vital substances and secondary metabolites Preliminary experiments were conducted for the in- from A. visnaga (L.) Lam which reveals the importance of vitro establishment and multiplication of microshoots and this valuable plant (Al-Snafi, 2013; Hashim et al., 2014). callus cultures. The best multiplication rate for the In addition to its traditional importance in preventing or microshoots was obtained in MS medium (Murashige and decreasing kidney stone formation in human patients, and Skoog, 1962) containing 0.5 mg/L BA in combination with its recent scientific importance against bronchial asthma 0.1 mg/L NAA, while the callus multiplication was best in and coronary diseases which are attributed to its essential MS medium containing 1.0 mg/L BA in combination with oils (Rose and Hulburd, 1992; Satrani et. al., 2004), Ammi 2.0 mg/L 2,4-D under light condition. To obtain the visnaga can be used as antitumoral (Beltagy and Beltagy, recommended mother stock for the present experiment, the 2015) and is considered as a good antifungal and microshoots (microshoot about 2.0 cm long and each flask antimicrobial agent against both gram-positive and gram- contained four microshoots) and callus clumps (clump negative bacteria (Rasooli et al., 2007; Dababneh, 2008; weight=0.5 g, with four clumps in each Petri dish) Ghareeb, et al., 2011; Khalfallah et al., 2011; Mahmood, underwent five subculturing intervals (four 2014; Sabry et al., 2014; Jaradat et al., 2015). weeks/interval). Accordingly, this study is conducted to investigate the antimicrobial potential of the in- vitro grown microshoots 2.3. Antimicrobial Effect and callus cultures of Ammi visnaga as they contain a wide 2.3.1. Crude Extraction array of chemical constitutes reported to be active against some strains of bacteria and fungi An aqueous extract of field and in-vitro grown plant material was prepared by stirring 4 g of previously dried 2. Material and Methods plant powder with 40 ml of sterilized distilled water, while 1 g of the dried plant powder was added to 10 mL 100 % of a methanol solvent for the methanolic extract 2.1. Khellin Test preparation. Both types of the extracts were left two days As mentioned earlier, khellin is a furanochromone and at 25°C with continuous shaking at the incubator shaker is exclusively extracted from two species which are Ammi adjusted at 200 rpm. This was followed by centrifugation visnaga (L.) Lam (Apiaceae) and Eranthis hyemalis L. at 2000 g for ten minutes and the resulted supernatant was (Ranunculaceae) (Kaul and Staba, 1967; El-Fiky, et al., concentrated by complete solvent evaporation under 1989). Due to the high similarity between Ammi visnaga vacuum using a rotary evaporator device, and was finally (L.) Lam and Ammi majus L seeds, the plant material was stored in sterile falcon tubes in a refrigerator at 4°C after identified by the taxonomist, Professor Ahmad Aloqla being dissolved with dimethyl sulphoxide (DMSO) to from the Biology Department at Yarmouk University, reach the 100 mg/ml stock for each extract and was Irbid, Jordan, using the Khellin test. This was very sterilized by a syringe filter with 0.45 μm pore size essential for the current research, as the tissue cultured (Jaradat et al., 2015). plant material (microshoots and callus) used in our 2.3.2. Antibacterial and Antifungal Activity of the Extracts experiments were started initially in-vitro from the seeds .
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