Effects of Culture Media and Explant Types on Callus and Shoot Induction of Annona muricata L.

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1 Malaya Journal of Biosciences 2014, 1(3): ISSN print / online Malaya Journal of Biosciences SHORT COMMUNICATION Open Access Full Text Article Effects of Culture Media and Explant Types on Callus and Shoot Induction of Annona muricata L. On-Usa Inwanna 1, Sainiya Samala 2, Charuvat Chanpradit 3, Natthaya Choosingh van Beem 3* 1 Master Degree Student of Biology, Faculty of Science, Thaksin University, Phatthalung 93110, Thailand 2 Department of Biology, Faculty of Science and Technology, Suratthani Rajabhat University, Suratthani 84100, Thailand 3,4 Department of Biology, Faculty of Science, Thaksin University, Phatthalung 93110, Thailand * For correspondence: natthaya@tsu.ac.th. Tel ext Article Info: Received 13 Oct 2014; Revised: 22 Nov 2014; Accepted 28 Nov ABSTRACT Annona muricata L. or Soursop (Dhurianthet, common name in Thai) is a member of Annonaceae growing in southern Thailand and in tropical climates throughout the world. To date, A. muricata L. has been more than 50 annonaceous acetogenins isolated from the stems, leaves and seeds. These secondary metabolites are evaluated for several studies include cytotoxicity, cell metabolism, cancer-associated protein/gene expression of lung, breast and cervix cancer cell lines. This study aimed to culture the explant types on callus and shoot of A. muricata L. in various media and growth hormones for increasing the secondary metabolite productions. Shoot regeneration (2-3 green leaves; average 2.0x4.3 cm) was showed at 60% in the formula including MS medium+0.5mg/l, BAP+0.05mg/l, NAA+0.8% and agar+2% sucrose. The most formation of compact callus (light green color; diameter was 1.50x1.20 cm) was showed at 86% in the medium of MS+0.2mg/l, BAP+0.2mg/l, NAA +0.8% and agar+2% sucrose. Keywords: Annona muricata L., Annonaceae, Tissue culture, Secondary metabolite production, Shoot regeneration. 1. INTRODUCTION Annona muricata L. belong to the family of Annonaceae. It has a widespread trough tropical distribution also as a native species in southern Thailand. A. muricata L. named as Soursop or Dhurian-thet, common name in Thai (Figure.1). Fruit of A. muricata L. is found to be edible and used commercially for the production of juice, candy, icecream and sherbets. Seed and leaves were used in the Copyright 2014 MJB traditional medicine to treat various diseases [1,2]. Many pharmacological studies report acetogenins, as secondary metabolites isolated from leaves and seed of this tree (Oberlies, Chang and McLaughlin. 1997: 6). Interestingly, the isolated acetogenin compounds display some of the pharmacological activities such as antitumoral, cytotoxicity, antiparasitic and pesticidal properties [3-5]. 155

2 Polyploidy induction of plant is a method for reforming plant to selective breeding. In general, polyploid plants have larger leaves and flowers than autopolyploid plants. Using Colchicine treatment under aseptic condition for increasing plant chromosome number to explore its secondary metabolite production has been used as standard protocol. Colchicine, a compound that effectively arrests mitosis at the anaphase stage, has been found to have a significant effect on polyploidy plant induction in vitro. Despite many reports of applying polyploidy techniques to plant by using microexplant in liquid media MS mixed with 2% (v/v) DMSO, BA 10 µm with % (w/v) Colchicine [6]. The present study aimed to evaluate the effects of culture media and explant types on callus and shoot induction of A. muricata L. under aseptic condition for acetogenins secondary metabolite productions. 2. MATERIALS AND METHODS 2.1 Young plant preparation and explant culture Fresh seeds of A. muricata L. from 5-year-old plants were collected in 2013 from Botanical Building, Department of Biology, Thaksin University. The experimental seeds were grown in a mixture containing 1:1:1 of sand rice, husk and coconut husk for 90 days. Shoots and petioles of seedlings were cut and cleaned in running tap water for 2 minutes. All plant parts were disinfected in 10% (w/v) HgCl 2 with Tween-20 for 10 min and thoroughly washed with sterile distilled water for 10 min. Shoots and petioles were germinated aseptically on half strength Murashige and Skoog (MS) medium (1962). Experimental design 5x2 Factorial in Completely Randomized Design (Factorial in CRD) was employed in order to test various treatment regimens encompassing 2 factors: explants and culture on five medium with growth regulators concentrations of Benzyladenine (BAP), Naphathaleneacetic (NAA) and 2,4-Dichlorophenoxy acetic (2,4-D). 2.2 Media composition Five medium with growth regulators as shown below, 1. MS1 (1/2 MS+2mg/l BAP 0.1mg/l NAA+0.8% agar+2% sucrose) 2. MS2 (1/2 MS+0.2mg/l BAP+0.2mg/l NAA +0.8% agar+2% sucrose) 3. MS3 (1/2 MS+0.5mg/l BAP+0.05mg/l NAA +0.8% agar+2% sucrose) 4. MS4 (1/2 MS+0.5mg/l NAA +0.8% agar+2% sucrose) 5. MS5 (1/2 MS+ 2mg/l 2, 4-D+0.8% agar+2% sucrose) Each part of plant was developed and inoculated at 25±2 C under cool white fluorescent of plant tissue culture room in 3 repeating bottles (30 pieces/bottle). Subculture propagated parts every 4 week to fresh media. 2.3 Data analysis Explants producing callus and shoot were observed and calculated by Analysis of Variance (ANOVA). Duncan s new multiple range test (DMRT) was tested to compare the differences of each group using The SAS system version RESULTS AND DISCUSSION Effects of culture media and explant types on callus and shoot induction of A. muricata L. under aseptic condition showed that all explants types effected to development to be callus or shoot because of the influence media with plant hormones as shown in Table 1 & 2 and Figure 2 & 3. Research was clearly showed the effects of culture media to callus when filling equal volume of auxin (NAA) and cytokinin (BAP). The appropriate concentrations of NAA and BAP on medium were effects to the development of various organs [7]. Increase in auxin induced growth of root but inhibited shoots. Shoot formation grew very slowly in MS2 and MS. Average of shoot tips on MS2, MS3 and MS5 media were difference (p<0.05), whereas MS1 and MS4 were non-difference. Possibly, the suitable combination of growth regulators for shoot induction was 8.9 µm (2 mg/l) BA and 0.54 µm (0.1 mg/l) NAA [8] by showing 4.8 shoots per explant cultured on MS and Nitsch medium, modified with 0.8% agar. Apparently, enhanced shoot elongation might be get from lower concentration hormones [9-11]. 4. CONCLUSION This study aimed to culture the explant types of A. muricata L., callus and shoot, in various media and growth hormones. Shoot regeneration (2-3 green leaves; average 2.0 x 4.3 cm.) was showed 60% in ½ MS medium+0.5mg/l BAP+0.05mg/l NAA +0.8% agar+2% sucrose. Formation of compacted callus (light green color) was showed 87%in ½ MS+0.2mg/l BAP+0.2mg/l NAA +0.8% agar+2% and average callus diameter was 1.50x1.20 cm. Although annonaceous induced explants requires BAP and NAA in culture media precisely, the explants provides a suitable different hormone additions. 156

3 Figure.3. components of Annona muricata L.: (A) Fruit; (B) Seedlings and (C) Flower. Table 1. Callus formation at 1 month of age from induced petioles in 5 treatment media Treatment Callus diameter Experimental results Survival rate (cm) (%) MS1 0.88x0.90 Compact callus (light green color) 60 MS2 1.50x1.20 Compact callus (light green color) 87 MS3 - Small callus 10 MS4 - Small callus (light color) 10 MS5 0.53x0.50 Brown Callus and very slow proliferation 20 Table 2. Shoot formation at 1 month of age from induced shoot tips in 5 treatment media Treatment New shoot per sample Experimental results Survival rate (%) Average of new shoot in each medium MS1 1-2 Small shoots, light color, leaves average 1.2x2.2 cm and very slow proliferation ±0.152 bc MS2 1 Yellow-green color and very slow proliferation b MS green new leave formation ±0.221 c (average 2.0x4.3 cm) MS4 1-2 green new leave formation ±0.163 bc (average 1.0x1.5 cm) MS5 1 Brown color shoot and very slow proliferation ±0.167 a All experiments were done in triplicates (p<0.05). All values are expressed as Mean±SD. 157

4 Figure 2. Induction of callus from petiole on the 1/2-Murashige and Skoog (MS) medium for 1 month Figure 3. Induction of shoot from tip on the 1/2-Murashige and Skoog (MS) medium for 1 month 158

5 Conflict of Interest The authors declare that they have no conflicts of interest. Acknowledgement The authors wish to thank for research fundings provided by Graduate School (Graduate School fund for Sciences) and Department of Biology, Faculty of Science (Scholarship for Potential Students), Thaksin University. References 1. Gajalakshmi S, Vijayalakshmi S and Devi RV (2012). Phytochemical and pharmacological properties of Annona muricata: A review. International Journal of Pharmacy and Pharmaceu-tical Sciences; 4(2); Salaeh A, Pimkot Y and van Beem CN (2013). Total phenolic compounds and cytotoxicity tests from Thurian-thet (Annona muricata L.) crude extracts. Proceeding, 23 th Thaksin University Annual Conference. pp Van Beem CN, Plansangket S, Nakleua C and Kongsong S (2012). Determination of total phenolic compounds of some extracted ethnophamacological plants against cancers, diabetes and allergies. Thaksin University Journal; 15(3); Tomo JR, Gallardo T, Aragon R, Cortes D, Estornell E (1999). Specific interactions of monotetrahydrofuranic Annonaceous acetogenins as inhibitor of mitochondrial complex I. Chemico- Biological Interactions; 122; Jackson BP and Rowson JM Alkaloid biogenesis in tetraploid Stramonium. Journal of Pharmacy and Pharmacology; 5; Jesus-Gonzalez LD and Weathers PJ (2003). Tetraploid Artemisia annua hairy root produce moreartemisinin than diploids. Plant Cel Report; 21; Oberlies NH, Chang CJ and McLaughlin JL (1997). Structure-activity relationships of diverse Annonaceous acetogenins against multidrug resistant human mammary adenocarcinoma (MCF-7/Adr) cells. Medicinal Chemistry; 40; Rasai, S, George, AP and Kantharajah AS (1995). Tissue culture of Annona spp. (cherimoya, atemoya, sugar apple and soursop): A review. Scientia Horticulturae; 62; Ye YM, Tong J, Shi XP, Yuan W and Li GR (2010). Morphological and cytological studies of diploid and colchichine-induced tetrapliod lines of carpe myrtle (Lagerstroemia indica L.). Scientia Horticulturae; 124; Benjoy, M and Harinharan M (1992). In vitro plantlet differentiation in Annona muricata. Micropropagation note; 31; Bouveier L, Guerif P, Djulbic M, Durel CE, Chevreau E, and Lespinasse Y (2002). Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers. Euphytica; 123;