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Journal of Fisheries science and Technology Special issue - 2015 CALLUS INDUCTION OF KAPPAPHYCUS ALVAREZII COLLECTED FROM KHANH HOA PROVINCE BY TISSUE CULTURE Khuc Thi An1, Van Hong Cam1 ABSTRACT Tissue culture techniques were applied for inducing callus of the red alga Kappaphycus alvarezii. The objective of this study was to obtain the optimal medium for callus induction from thallus explants of Kappaphycus alvarezii. Seaweed was collected from the Van Phong Bay (Khanh Hoa Province) and then was acclimatized in greenhouse and in semi-sterile culture in the laboratory. Sterilized explants were cultured on PES, ESS and CW media solidified with 1.5% Bacto Agar. The result indicated that the optimal medium for callus induction was PES solidified medium. In the PES medium, combinations of plant growth regulators i.e. BAP, IAA and NAA were added. Types of callus formed were: filamentous callus and compact callus. Time and percentage of callus formation were different depending on the concentration, type of phytohormone, and type of callus as well. In particular, BAP 1 mg/l + IAA 5 mg/l induced fillamentous callus with 63.3% but no compact callus induced after 22 days while BAP 0 mg/l + NAA 0.5 mg/l induced 43.3% fillamentous callus and 10% of compact callus after 19 days. Our results can be the premise for next researches on multiplying selected strains and increasing seed stocks production of Kappaphycus alvarezii. Keywords: Kappaphycus alvarezii, tissue culture, callus, seaweed, plant growth hormones I. INTRODUCTION Kappaphycus alvarezii is an important source of the industrial gel carrageenan. It is one of the important commercial species cultivated in Southeast Asia. In Vietnam, Kappaphycus alvarezii is cultured in the Central of Vietnam. Many places in Khanh Hoa province grow this commercial species. However, dwindling resources of seedstocks, loss of genetic variability, and fastspreading diseases has significant negative impacts on the carrageenophyte seaweed industry. One approach developed to ease these effects is the optimization of tissue culture techniques to produce a large quantity of high quality Kappahycus alvarezii seedstock for nursery purposes. Successful micro-propagation of K. alvarezii has been reported by Dawes et al. (1993), Hurtado and Biter (2007), and Hayashi et al. (2009) 1 Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and develop experimental system for in vitro culture. Generally, seaweed tissue culture stages include preparation of axenic explants, callus induction and regeneration of callus into thallus and young plantlets of seaweed (Hayashi et al., (2009). The objective of this study was to obtain the optimal medium for callus induction from thallus explants of which were collected from Khanh Hoa province. II. MATERIALS AND METHODS 1. Sample transport Clean and healthy plants of Kappaphycus alvarezii (reddish brown) was collected from commercial farms in Van Phong Bay ((12.6o N, Institute of Biotechnology and Environment – Nha Trang University; Email: ankhucthi@yahoo.com 44 • NHA TRANG UNIVERSITY Journal of Fisheries science and Technology Special issue - 2015 109.3o E) and brought to laboratory under cool sterilization methods modified from Hurtado and temperature (22 ± 2 C) and with a quite wet Biter (2007). Briefly, selected apical segments, condition (Sulistiani et al., 2012). approximately 5 cm long were cut using a o sterile blade, rinsed 3–4 times in autoclaved 2. Seaweed acclimatization After arrival, the branches of seaweed were trimmed to about 10 cm and cleaned thoroughly in filtered seawater with a soft brush to remove dirty attached (Reddy et al., 2003). To reduce mortality at the time of semi-sterile culture in the laboratory, new seaweed need to be acclimatized and maintained in a clean and controlled environment before the semi-sterile culture in the laboratory. The seaweed was cultured in sea-water in small containers with different shapes: globular, triangle, rectangular. The branches were used for initiation of unialgal cultures and for acclimatization to laboratory conditions by growing in Provasoli enriched seawater/ PES medium (Provasoli, 1968), modified Erd Schreibers seawater/ESS (Suto, 1959) and Conwy/CW medium (Liao et al., 1983) and seawater. The segments then were saken in A1 antibiotic solution (in our laboratory) mixture with different time (0, 10, 30 mins, 1, 2, 6, 12, 24, 48 hours). During antibiotic treatment, the cultures were maintained as static cultures under similar conditions as described for acclimatization except the light intensity which was at 500 lux (≈10-15 µmol photons/m2/s). The segment then was rinsed 3 – 4 times with autoclaved seawater to eliminate antibiotics, and then was wiped gently with sterile filter papers (Whatman no. 1, Maidstone, UK) before placed in 24-well culture plates containing PES and incubated at 23 - 25°C, 12:12h light:dark cycle and 10–15 µmol photons/m2/s light intensity. There were 30 samples for each treatment. The mortality rate and contamination rate were read after 14 days. seawater/SW as control. To eliminate diatom 4. Effects of culture medium and sample growth, GeO2 (10 mg/L) was added to all size on callus induction culture media during first 2 weeks of culture The media PES, ESS, CW and SW added (Reddy et al., 2003). Salinity was adjusted by 1.5% agar were used to test the effect of daily to maintain at 32 psu. During the culture media on callus and shoot regeneration. acclimatization period, algae were continuously Healthy acclimated seaweed were cut into aerated and maintained at 22 ± 2 C under cool small pieces (1 – 5mm) for culturing. In addition, white fluorescent tube lights at 2500 lux (≈35 three sample sizes were tested: 1 mm, 3 mm, µmol photons/m /s) with a 12:12-h light : dark 4-5 mm - modifed from Muñoz et al. (2006). cycle. The experiment was repeated 3 times. Each treatment was done with 30 samples. After 35 days, survial rate of seaweed (%) and The survial rate and callus regeneration rate daily growth rate (%) was calculated (Hurtado were read after 5 weeks. o 2 et al., 2001). 3. Axenic cultures 5. Effects of phytohormones on callus induction After being acclimatized, healthy apical Explants (4 – 5 cm in PES medium) that segments were selected for tissue culture. had been observed for 2 weeks and did Axenic explants were established by surface not contaminate were used as explants for NHA TRANG UNIVERSITY • 45 Journal of Fisheries science and Technology Special issue - 2015 callus induction. Explants were cut into pieces It was characterized by the formation of buds with the length of 4-5 mm. Each explant then at the cuts at the tip of thallus (Fig. 1) was wiped gently with sterile tissue papers to Differences were obtained between the remove moisture and mucilaginous substances survival rate and daily growth rate (P<0.001) on from the cut ends. Explants were then PES, ESS, CW media, and globular, triangle, planted on treatment media for callus induction.To rectangular tanks. Among 3 different media and investigate the optimal culture media, explants 3 different shapes of container, PES medium were cultured on PES medium solidified with and globular tank got the highest survival rate 1.5% Bacto Agar. Plant growth regulators were and daily growth rate (Fig. 2). used: BAP, NAA and IAA which were utilized separatedly or together. The concentration of phytohormones were 0, 0.5, 1 mg/L with BAP and NAA, 0, 2.5, 5 mg/L with IAA (Sulistiani và cs. 2012). Each treatment was done with 30 samples. The cultures were stored in a culture room, The cultures were illuminated with fluorescent lamps at 1500 lux of light intensity with a 12 : 12 light and dark cycle. 6. Statistical analysis Data were analyzed statistically using Analysis of Variance (one-way or two-way ANOVA) using Microsoft excel. III. RESULTS AND DISCUSSION 1. Seaweed acclimatization The highest mortality of K. Alvarezii samples occurred in the first two weeks. From the 2. Axenic cultures Survival rate got 90% and contamination rate got 0% at 1 hour sterilization. There were significant different between experimental groups (One-way ANOVA, P<0.05). The results showed that our protocol costs less in terms of time, and chemicals compared to other publications (Hurtado and Biter, 2007; Reddy et al., 2003). 3. Effects of culture medium and sample size on callus induction There was no callus developed in control group (seawater) while CW medium gave the highest mortality (90%) in the tissue culture. PES was the best culture medium that gave highest survival rate (93%) and callus induction rate (53%) (Two-way ANOVA, P < 0.005). 3 week, seaweeds started to be apdapted to Sample size with 4-5mm was suitable for laboratory conditions. After 35 days maintained callus induction (47%) and survival rate (87%) in greenhouse, the tip of thallus began to grow. (P < 0.005). rd Figure 1. Bud formation at the cut and tip of thalus 46 • NHA TRANG UNIVERSITY Journal of Fisheries science and Technology Figure 2. Kappaphycus alvarezii in globular tank Special issue - 2015 4. Effects of phytohormones on callus induction There are two types of callus formed including filamentous callus (Fig. 3) and compact callus (Fig. 4). Time and percentage of callus formation were different depending on the concentration, type of plant growth hormone, and type of callus as well. i.e: BAP 1 mg/l + IAA 5 mg/l induced fillamentous callus with 63.3% but no compact callus after 22 days while BAP 0 mg/l + NAA 0.5 mg/l induced 43.3% fillamentous callus and 10% of compact callus after 19 days (Table 1). In the control group (PES without phytohormone), there was no compact callus but fillamentous callus and buds (Fig. 5). Those results were somehow simillar to previous publications. Hayashi et al. (2009) proposed that the ratio 5IAA:1BAP could induce the highest fillamentous callus (same with our result). Callus in other publications were also formed with low proportion (Hayashi et al., 2009; Muñoz et al., 2006). Figure 3. Growth of fillamen clumps on fillametous in PES medium added by BAP 1 mg/l + IAA 5 mg/l (a). Cross section of fillamentous callus at magnification 40X (b), 400X (c) and 1000X (d) NHA TRANG UNIVERSITY • 47 Journal of Fisheries science and Technology Special issue - 2015 Figure 4. Compact callus at the edge of the cut (a, b). Cross section of compact callus at 400X (c) and 1000X (d) magnification Figure 5. Buds formation of Kappaphycus alvarezii tissue culture 48 • NHA TRANG UNIVERSITY Journal of Fisheries science and Technology Table 1. Effect of phytohormone concentration on the growth of callus after 6-week planting Ratio of formation (%) Concentration of BAP/IAA/NAA (mg/l) Buds Fillamentous callus Compact callus Special issue - 2015 IV. CONCLUSIONS Callus of Kappaphycus alvarezii were successfully induced. Kappaphycus alvarezii brought from Van Phong Bay to Nha Trang University was acclimatized in laboratory condition (PES medium, 32% salinity, 22 ± 2oC 0/0/0 23.3 46.7 0 0/2.5/0 33.3 20 0 0/5/0 0 26.7 0 0/0/0.5 0 43.3 10 0/0/1 0 10 0 0.5/0/0 0 40 0 0.5/2.5/0 10 20 0 0.5/5/0 13 50 0 0.5/0/0.5 0 56.7 0 0.5/0/1 0 0 3 1/0/0 0 53.3 0 compact callus formation. 1/2.5/0 0 16.7 3 01/05/00 0 63.3 0 ACKNOWLEDGMENTS 1/0/0.5 0 60 6.7 1/0/1 0 0 0 under cool white fluorescent tube lights at 2500 lux, with a 12:12-h light:dark cycle) for 35 days. The procedure for sterilizing the explant cost less time, steps and chemicals than other researches. The best medium for Kappaphycus alvarezii tissue culture and callus induction was PES with 1mg/l BAP + 5mg/l IAA or 1mg/l BAP + 5mg/l NAA (for fillamentous callus) while PES with 0.5 mg/l NAA could stimulate This research was contributed by Raul Rincones (Consultor Indepeniente) and Viet Intelligence Corporation. REFERENCES Dawes C. J., Trono G. C., Jr., Lluisma A. O. (1993) Clonal propagation of Eucheuma denticulatum and Kappaphycus alvarezii for Philippine seaweed farms. Hydrobiologia 260-261(1): 379-383 2. Hayashi L., Yokoya N., Kikuchi D., Oliveira E. (2009) Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae). In: Borowitzka M., Critchley A., Kraan S., Peters A., Sjøtun K., Notoya M. (eds) Nineteenth International Seaweed Symposium. Springer Netherlands, pp 203-209 3. Hurtado A. Q., Agbayani R. F., Sanares R., de Castro-Mallare M. T. R. (2001) The seasonality and economic feasibility of cultivating Kappaphycus alvarezii in Panagatan Cays, Caluya, Antique, Philippines. Aquaculture 199(3–4): 295-310 4. Hurtado A. Q., Biter A. B. (2007) Plantlet regeneration of Kappaphycus alvarezii var. adik-adik by tissue culture. J Appl Phycol 19(6): 783-786 5. Liao I. C., Su H. M., Lin J. H. (1983) Larval foods for penaeid prawn. In: Mc Vey JP, editor. CRC Handbook of Mariculture volume 1: Crustacean Aquaculture. CRCPress, Inc. Boca Raton, Florida: 29-60 6. Muñoz J., Cahue-López A., Patiño R., Robledo D. (2006) Use of plant growth regulators in micropropagation of Kappaphycus alvarezii (Doty) in airlift bioreactors. J Appl Phycol 18(2): 209-218 7. Provasoli L. (1968) Media and prospects for the cultivation of marine algae. In Watanabe, A. & Hattore, A. [Eds.] Culture and Collection of Algae. Japanese Society of Plant Physiology: 63–75 8. Reddy C. R. K., Kumar G. R. K., Siddhanta A. K., Tewari A., Eswaran K. (2003) In vitro somatic embryogenesis and regeneration of somatic embryos from pigmented callus of Kappaphycus alvarezii (Doty) Doty (Rhodophyta, Gigartinales). Journal of Phycology 39(3): 610-616 9. Sulistiani E., Soelistyowati D. T., Alimuddin, Yani S. (2012) Callus induction and filaments regeneration from callus of cottonii seaweed Kappaphycus alvarezii (Doty) collected from Natuna Islands, Riau Islands province. Journal of Botropia 19(2): 103-114 10. Suto S. (1959) Skeletonema no tame no jinkou baiyoueki. SuisanZouskoku 7: 17 - 19 [in Japanese] 1. NHA TRANG UNIVERSITY • 49