Tubers were used as explants in in vitro mass propagation of Rodent Tuber (Typhonium flagelliforme). The explants were obtained from sterile plantlets and placed in shoot induction medium containing basal salts of Murashige and Skoog (MS) and various concentrations of 6-benzylaminopurine (BAP) and -naphthaleneacetic acid (NAA). Treatment containing 5 mg/l (w/v) of BAP with 1 mg/l (w/v) of NAA produced the highest number of shoots per explant (29.17) after 12 weeks of culture and also the highest mean fresh weight of shoots formed in treatment containing 5 mg/l (w/v) of BAP with 1 mg/l (w/v) of NAA. For ex vitro establishment, well- rooted plantlets were transferred in potting medium containing peatmoss, perlite and vermiculite (3:1:1)

Kadir, MA

Nobakht, GM

Stanslas, J

English

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African Journal of Biotechnology Vol. 8 (24), pp. 6840-6843, 15 December, 2009     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals      Full Length Research Paper  In vitro mass propagation of Typhonium flagelliforme as affected by plant growth regulators  Nobakht, G. M.1, Kadir, M. A.1* and Stanslas, J.2  1Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. 2Department of Medicine, Faculty of Medicine and Health Science, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysia.  Accepted 5 October, 2009  Tubers were used as explants in in vitro mass propagation of Rodent Tuber (Typhonium flagelliforme). The explants were obtained from sterile plantlets and placed in shoot induction medium containing basal salts of Murashige and Skoog (MS) and various concentrations of 6-benzylaminopurine (BAP) and -naphthaleneacetic acid (NAA). Treatment containing 5 mg/l (w/v) of BAP with 1 mg/l (w/v) of NAA produced the highest number of shoots per explant (29.17) after 12 weeks of culture and also the highest mean fresh weight of shoots formed in treatment containing 5 mg/l (w/v) of BAP with 1 mg/l (w/v) of NAA. For ex vitro establishment, well- rooted plantlets were transferred in potting medium containing peatmoss, perlite and vermiculite (3:1:1).   Key words: Typhonium, 6-benzylaminopurine, -naphthaleneacetic acid, indole-3-butyric acid, in vitro culture, mass propagation.   INTRODUCTION  Typhonium flagelliforme is a medicinal herb which belongs to the Araceae (Arum) family. It can be found from India to Australia and is spread northward to the sub-temperate areas of the Eastern Asia up to Sri Lanka (Nicolson and Sivadasan, 1981). In a report by World Health Organization, it was acclaimed that a high percen-tage of the world’s population are using herbal medicine as drug and there is a growing interest in the use of traditional medicines (Tilburt and Kaptchuk, 2008). How-ever, herbal medicines, like other natural resources, have very limited sources. Thus, artificial regeneration of her-bal plants becomes important. Plant tissue culture system offers a tool for a large scale production of genetically similar plants (Wawrosch et al., 1999). Perry and Metzger (1980) reported that the people in the Philippines have been using the flowers of T. flagelliforme to arrest bleed-ing and as remedial for the treatment of injury. In addition another species of Typhonium, T. divaricatum is used in     *Corresponding author. E-mail: mihdzar@agri.upm.edu.my or motaharenobakht@yahoo.com. Tel: +603_8946 4114. Fax: +603-8946 4146. China for curing of internal injuries and oedema. Another report by Su et al. (2000) also showed that the rhizomes extract of T. divaricatum has been used for the treatment of therapeutic coughs and pulmonary conditions. Chan et al. (2000) studied the different media for deve-loping the tissue culture tecnique of T. flagelliforme. They discovered that among Murashige and Skoog (MS) (Murashige and Skoog, 1962), Nitsch and Nitsch (NN), Gamborg B5 (GB5) and White (W) medium, MS was the most suitable medium for this particular plant. The best medium for maximum shoot number, with normal complete plant, was reported on MS medium supplemented with 0.3 mg/l BA and 0.5 mg/l Indole-3- butyric acid (IBA). In 2003, Koh and Chan published a paper on T. flagelliforme micropropagation in immersion culture. In their study, they used 5 L fermentor containing liquid modified MS medium supplemented with 4% sucrose, 1.33 µmol/l BA and 2.46 µmol/l IBA. The problems related to T. flagelliforme are sensitivity to specific growth condition in the natural environment such as moist and be in the shady area which provided the biggest need to produce the plant in vitro in massive amount. This paper reports the effect of BAP either  alone  or  in  Nobakht et al.         6841     abcdbcdcdcdbcdcddeebcdbc bc05101520253035BAP0 NAA0BAP0 NAA0.5BAP0NAA1BAP1NAA0BAP1NAA0.5BAP1NAA1BAP5NAA0BAP5NAA0.5BAP5NAA1BAP10NAA0BAP10NAA0.5BAP10NAA1Combination of BAP and NAA(mg/l)Number of shoots formed per explantcdbcbcdabbcdcdcdcdcddee05101520253035BAP0NAA0BAP0NAA0.5BAP0NAA1BAP1NAA0BAP1NAA0.5BAP1NAA1BAP5NAA0BAP5NAA0.5BAP5NAA1BAP10NAA0BAP10NAA0.5BAP10NAA1Mean shoot weight(g)                                                                    A B   Combination of BAP and NAA (mg/l)   Figure 1. The effect of BAP, in combination with NAA, on mean number of shoots (A) and on mean weight of shoots (B) produced per explant after 12 weeks of culture. Means followed by the same letter(s) are not significantly different using DNMRT at  = 0.05.    combination with NAA, on adventitious shoot proliferation from tuber explants for mass production of T. flagella-forme.   MATERIALS AND METHODS  Plant material  The tuber explants were obtained from in vitro established T. flagel-liforme plantlets and then cultured on proliferation medium containing complete MS salts supplement with 30 g/l sucrose.   Medium composition and treatment  The explants were cultured on MS medium containing different concentration and combination of BAP (0, 1, 5, 10 mg/l) and NAA (0, 0.5, 1 mg/l) for shoot induction. The pH of the medium was adjus-ted to 5.7 and MS medium without any plant growth regulator consider being the control of experiments.    Parameters recorded  The parameters taken in this study were the mean number of shoots per explant and mean fresh weight of shoots produced per explant. Data were collected every four weeks until the 12th week of culture whereas the growth condition was observed every week.   Experimental design and statistical analysis   The experiment was conducted in a Randomized Complete Block Design (RCBD). Data were analyzed using the analysis of variance (ANOVA) and Duncan New Multiple Range Test (DNMRT) at = 5% was applied for comparison between the treatment means.  6842         Afr. J. Biotechnol.      Figure 2. Shoot production, after 2(A), 4(B), 8(C) and 12(D) weeks of culture in BAP5NAA1 mgl-1. Bar = 1 cm.    RESULTS AND DISCUSSION  Within two to three weeks of culture on the shoot induc-tion medium, new shoots started to proliferate from the tuber (Figure 2A). For this plant the tuber has the main role in the propagation process. The treatment combinations and concentrations of BAP and NAA tested were found to affect the mean number of shoots produced per explant. A high response was also detected from 9 weeks of culture until 12 weeks, while significant differences were observed among the treat-ments containing different combinations in terms of the number of shoots formed (Figure 1A) as well as fresh weight per explant (Figure 1B). Treatments containing 5 mg/l (w/v) of BAP with 1 mg/l (w/v) of NAA (BAP5NAA1) produced the highest number of shoots per explant (29.17) after 12 weeks of culture (Figure 1A). The multiple shoots developed normally as shown in Figure 2. According to Chan et al. (2000), MS medium with low concentration of BAP and NAA could cause abnormal and incomplete plantlet formation. They also found that the best medium which enabled the buds of T. flagella-forme to produce the most shoots was treatment with 0.5 mg/ l NAA and 0.5 mg/ l BAP, although the shoots pro-duced were abnormal with twisted leaves and no roots. Treatments containing BAP, either alone or in combi-nation with NAA significantly increased the mean shoot weight formed per explant as compared to the control (BAP0NAA0), Treatments containing different combi-nations of BAP and NAA indicated no significant diffe-rence among them on mean shoot weight obtained per explant. Treatment with 5 mg/l (w/v) BAP and 1 mg/l (w/v) NAA (B5N1) produced the highest mean fresh weight of ex-plant (Figure 1B).Treatments containing 0 mg/l(w/v) NAA in combination with 0 to 10 mg/l (w/v) BAP caused no significant differences in mean fresh weight per explant. The highest concentration of BAP at 10 mg/l (w/v) either alone or in combination with NAA 0 to 1 mg/l (w/v) (BAP10NAA0, BAP10NAA0.5, BAP10NAA1) did not differ significantly among them in terms of mean number of shoots per explant and mean shoot weight. However they were significantly different when compared to  the  control      (BAP0NAA0). Rooted shoots produced simultaneously in the shoots induction medium, they were successfully acclimatized in potting medium containing peatmoss, perlite and vermin-culite (3:1:1) and grew in the natural environment. The ratio of survival plants was 90% and no observable differences appeared among the transferred.   Conclusion   The results indicated that tubers are potential explants for in vitro mass propagation of Typhonium flagelliforme. BAP alone or in combination with NAA was more effective on enhancing shoot proliferation and mean fresh weight of explants. Combination of 5 mg/l BAP and 1 mg/l NAA was selected as the most suitable concentration for the shoot initiation and multiplication as well as producing the highest mean fresh weight of shoots. Regenerated plants survived and grew normally in natural environ-ment. The process, developed in this study, is suggested for rapid and efficient in vitro mass propagation of T. flagelliforme.                                           Nobakht et al.         6843    REFERENCES  Chan LK, Su TS, Pargini N, Teo CKH (2000). In vitro propagation of Typhonium flagelliforme. In Vitro Cell. Dev. Biol. 36: 402-406. Koh WY, Chan LK (2003) Micropropagation of Typhonium flagelliforme in immersion cultures. J. Trop. Med. Plants 4: 91-96.4. Murashige T, Skoog  F (1962). A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15: 473-497. Nicolson DH, Sivadasan M (1981). Four frequently confused species of Typhonium schott (Araceae). Blumea, 27: 483-497. Perry MP, Metzger J (1980). Mediconal plants of East and Southeast Asia. Cambridge, Massachusetts, USA: MIT Press, p. 24. Su TS, Chan LK, Teo CKH (2000). The morphological studies of Typhonium species found in Malaysian forest. Ann. Microscopy, 1: 55-63. Tilburt JC, Kaptchuk TJ (2008). Herbal medicine research and global health: an ethical analysis. Bulletin of the World Health Organization, 86: 594-599. Wawrosch C, Maskay N, Kopp B (1999). Micropropagation of the threatened Nepalese medicinal plant Swertia chirata Buch. Ham Ex Wall. Plant Cell Rep. 18: 997-1001.   

In vitro mass propagation of Typhonium flagelliforme as affected by plant growth regulators

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In vitro mass propagation of Typhonium flagelliforme as affected by plant growth regulators

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