Sains Malaysiana 45(5)(2016): 795–802

A Medicinal Ginger, Boesenbergia rotunda: From Cell Suspension Cultures to Protoplast Derived Callus

(Halia perubatan, Boesenbergia rotunda: Daripada Kultur Sel Penggantungan kepada Kalus daripada Protoplas)

HAO-CHEAK TAN¹, BOON-CHIN TAN², SHER-MING WONG¹ & NORZULAANI KHALID^1,2*

¹Institute of Biological Sciences, Faculty of Science, University of Malaya, Lembah Pantai,

50603 Kuala Lumpur, Malaysia

²Centre for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia

Diserahkan: 26 September 2014/Diterima: 20 November 2015

ABSTRACT

Boesenbergia rotunda is a medicinal ginger that has been found to contain several bioactive compounds such as boesenbergin A, panduratin A, cardamonin, pinostrobin and pinocembrin. These compounds are useful in treating various ailments, such as oral diseases, inflammation and have also been used as an aphrodisiac. In this study, an efficient protocol for developing and isolating protoplast cultures for B. rotunda has been established. Rhizome buds of B. rotunda were used as explants to initiate callus growth and the established cell suspension cultures were used to optimize their growth conditions. Our results indicated that embryogenic suspension cultures in liquid Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose produced the highest growth rate (μ = 0.1125), whereas no promotive effect was seen in the presence of 2,4-dichlorophenoxyacetic acid and those that underwent sonication treatment. Amount of protoplasts isolated ranging from 1-5 × 105 protoplast per mL were isolated using 0.25% (w/v) macerozyme and 1% (w/v) cellulase for 24 h under continuous agitation (50 rpm) in dark condition. Of the isolated protoplasts, 54.93% were viable according to fluorescein diacetate staining test. Micro-colonies were recovered in liquid MS medium containing 9 g/L mannitol, 2 mg/L 1-naphthaleneacetic acid and 0.5 mg/L benzylaminopurine (BAP) for 4 weeks and subsequently transferred to solid MS medium supplemented with 0.5 mg/L BAP for callus initiation. The protoplast system established in this study would be useful for genetic manipulation and modern breeding program of  B. rotunda.

Keywords: Cell suspension culture; medicinal ginger; micropropagation; protoplasts

ABSTRAK

Boesenbergia rotunda ialah halia ubatan yang didapati mengandungi beberapa sebatian bioaktif seperti boesenbergi A, panduratin A, cardamonin, pinostrobin dan pinocembrin. Sebatian ini berguna dalam merawat pelbagai penyakit seperti penyakit mulut, keradangan dan juga telah digunakan sebagai afrodisiak. Dalam kajian ini, satu protokol berkesan untuk membangun dan mengasingkan budaya protoplas untuk B. rotunda telah dibentuk. Tunas rizom B. rotunda telah digunakan sebagai eksplan untuk memulakan pertumbuhan kalus dan kultur penggantungan sel yang telah dibentuk digunakan untuk mengoptimumkan keadaan pertumbuhan mereka. Hasil kajian kami menunjukkan bahawa kultur penggantungan embriogenik dalam medium cecair Murashige dan Skoog (MS) ditambah dengan 3% (w/v) sukrosa menghasilkan kadar pertumbuhan yang paling tinggi (μ = 0.1125), manakala tiada kesan penggalakan dilihat dengan kehadiran asid 2,4-dichlorophenoxyacetic dan orang-orang yang menjalani rawatan sonikasi. Jumlah protoplas yang diasingkan adalah antara 1-5 × 10⁵ setiap mL telah diasingkan menggunakan 0.25% (w/v) maserozim dan 1% (w/v) selulase untuk 24 h bawah penggoncangan berterusan (50 rpm) dalam keadaan gelap. Daripada pencilan protoplas, 54.93% adalah berdaya maju mengikut ujian pewarnaan fluoresein diasetat. Micro-koloni ditemui dalam medium cecair MS yang mengandungi 9 g/L manitol, 2 mg/L 1-naftalenaasetik asid dan 0.5 mg/L benzylaminopurine (BAP) selama 4 minggu dan kemudiannya dipindahkan kepada medium pepejal MS ditambah dengan 0.5 mg/L BAP untuk permulaan kalus. Sistem protoplas yang dibentuk dalam kajian ini akan berguna untuk manipulasi genetik dan program pembiakan moden B. rotunda.

Kata kunci: Halia ubatan; kultur penggantungan sel; mikrorambatan; protoplas

RUJUKAN

Abdullah, M.A., Ali, A.M., Marziah, M., Lajis, N.H. & Ariff, A.B. 1998. Establishment of cell suspension cultures of Morinda elliptica for the production of anthraquinones. Plant Cell Tissue and Organ Culture 54: 173-182.

Al-Khayri, J.M. 2012. Determination of the date palm cell suspension growth curve, optimum plating efficiency, and influence of liquid medium on somatic embryogenesis. Emirates Journal of Food and Agriculture 24: 444-455.

Aoyagi, H. 2011. Application of plant protoplasts for the production of useful metabolites. Biochemical Engineering Journal 56: 1-8.

Bohm, H., Anthony, P., Davey, M.R., Briarty, L.G., Power, J.B., Lowe, K.C., Benes, E. & Groschl, M. 2000. Viability of plant cell suspensions exposed to homogeneous ultrasonic fields of different energy density and wave type. Ultrasonics 38: 629-632.

Chen, L.C.M., Craigie, J.S. & Xie, Z.K. 1994. Protoplast production from Porphyra linearis a simplified agarase procedure capable of commercial application. Journal of Applied Phycology 6: 635-639.

Davey, M.R., Anthony, P., Power, J.B. & Lowe, K.C. 2005. Plant protoplasts: Status and biotechnological perspectives. Biotechnology Advances 23: 131-171.

Fitzsimons, P.J. & Weyers, J.D.B. 1985. Properties of some enzymes used for protoplast isolation. In The Physiological Properties of Plant Protoplasts (Proceeding in Life Science), edited by Pilet, P.E. Springer-Verlag. pp. 12-23.

Geetha, S.P., Babu, K.N., Rema, J., Ravindran, P.N. & Peter, K.V. 2000. Isolation of protoplasts from cardamom (Elettaria cardamomum Maton.) and ginger (Zingiber officinale Rosc.). Journal of Spices and Aromatic Crops 9: 23-30.

Grosser, J.W. & Gmitter, F.G. Jr. 2011. Protocol fusion for production of tetraploid and triploid: Application for scion and rootstock breeding in citrus. Plant Cell Tissue and Organ Culture 104: 343-357.

Grzebelus, E., Szklarczyk, M. & Baranski, R. 2011. An improved protocol for plant regeneration from leaf– and hypocotyl– derived protoplasts of carrot. Plant Cell, Tissue and Organ Culture 109(1): 101-109.

Ho, H.Y., Liang, K.Y., Lin, W.C., Kitanaka, S. & Wu, J.B. 2010. Regulation and improvement of triterpene formation in plant cultured cells of Eriobotrya japonica Lindl. Journal of Bioscience and Bioengineering 110: 588-592.

Huang, H.Y., Wang, Z.Y., Cheng, J.T., Zhao, W.C., Li, X., Wang, H.Y., Zhang, Z.X. & Sui, X.L. 2013. An efficient cucumber (Cucumis sativus L.) protoplast isolation and transient expression system. Scientia Horticulturae 150: 206-212.

Jiang, F., Zhu, J. & Liu, H.L. 2013. Protoplasts: A useful research system for plant cell biology, especially dedifferentiation. Protoplasma 250: 1231-1238.

Jumin, H.B. 2013. Somatic embryogenesis via protoplast cultures of Fortunella hindsii (Champ.) and their regeneration into precocious flowering plants. Journal of Agricultural Technology 9(4): 999-1012.

Khatri, A., Dahot, M.U., Khan, I.A. & Nizamani, G.S. 2010. An efficient method of protoplast isolation in banana (Musa spp.). Pakistan Journal of Botany 42(2): 1267-1271.

Kirchhoff, J., Raven, N., Boes, A., Roberts, J.L., Russell, S., Treffenfeldt, W., Fischer, R., Schinkel, H., Schiermeyer, A. & Schillberg, S. 2012. Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting. Plant Biotechnology Journal 10: 936-944.

Lee, E.J., Mobin, M., Hahn, J.E. & Paek, K.Y. 2006. Effects of sucrose, inoculum density, auxin, and aeration volume on cell growth of Gymnema sylvestre. Journal of Plant Biology 49: 427-431.

Leguay, J.J. & Guern, J. 1975. Quantitative effects of 2,4-dichlorophenoxyacetic acid on growth of suspension-cultured Acer pseudoplatanus cells. Plant Physiology 56: 356-359.

Miller, M.W., Miller, D.L. & Brayman, A.A. 1996. A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound in Medicine and Biology 22: 1131-1154.

Murashige, T. & Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.

Ochatt, S.J. & Power, J.B. 1992. Plant regeneration from cultured protoplasts of higher plants. In Comprehensive Biotechnology Second Supplement, edited by Moo-Young, M., Warren, G.S. & Fowler, MW. New York: Pergamon Press. pp. 99-127.

Panda, A.K., Mishra, S. & Bisaria, V.S. 1992. Alkaloid production by plant cell suspension cultures of Holarrhena antidysenterica: I. Effect of major nutrients. Biotechnology and Bioengineering 39: 1043-1051.

Park, J.K., Park, S. & Craven, J.E. 2012. Protoplast Isolation and Fusion, Techniques and Experiments in Plant Tissue Culture. 3rd ed. Salt Lake City: Academic Press. pp.147-152.

Patel, H. & Krishnamurthy, R. 2013. Elicitors in plant tissue culture. Journal of Pharmacognosy and Phytochemistry 2(2): 60-65.

Pati, P.K., Sharma, M. & Ahuja, P.S. 2005. Extra thin alginate film: An efficient technique for protoplast culture. Protoplasma 226: 217-221.

Patil, R.S., Davey, M.R., Power, J.B. & Cocking, E.C. 2003. Development of long-term suspension cultures of wild tomato species, Lycopersicon chilense Dun. as regular source of protoplast: An efficient protoplast-to-plant system. Indian Journal of Biotechnology 2: 504-511.

Pindel, A. 2007. Optimization of isolation condition of Cymbidium protoplasts. Folia Horticulturae 19: 79-88.

Rao, R.S. & Ravishankar, G.A. 2002. Plant cell cultures: Chemical factories of secondary metabolites. Biotechnology Advances 20: 101-153.

Ratanasanobon, K. & Seaton, K.A. 2013. Protoplast isolation for species in the Chamelaucium group and the effect of antioxidant enzymes (superoxide dismutase and catalase) on protoplast viability. In Vitro Cellular and Developmental Biology – Plant 49: 593-598.

Reusink, A.W. & Thimann, K.V. 1965. Protoplasts from the Avena coleoptile. Botany 54: 56-64.

Rezazadeh, R. & Niedz, R.P. 2015. Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design. Plant Cell, Tissue and Organ Culture 122(3): 585-604.

Schenk, R.U. & Hildebrant, A.C. 1969. Production of protoplasts from plant cells in liquid culture using purified commercial cellulases. Crop Science 9: 629-631.

Sudwan, P., Saenphet, K., Aritajat, S. & Sitasuwan, N. 2007. Effects of Boesenbergia rotunda (L.) Mansf. on sexual behaviour of male rats. Asian Journal of Andrology 9: 849- 855.

Tan, B.C., Tan, S.K., Wong, S.M., Ata, N., Rahman N.A. & Khalid, N. 2015. Distribution of flavonoids and cyclohexenyl chalcone derivatives in conventional propagated and in vitro-derived field-grown Boesenbergia rotunda (L.) Mansf. Evidence-based Complementary and Alternative Medicine 2015: Article ID 451870.

Tan, E.C., Lee, Y.K., Chee, C.F., Heh, C.H., Wong, S.M., Thio, C.L.P., Foo, G.T., Khalid, N., Abdul Rahman, N., Karsani, S.A., Othman, S., Othman, R. & Yusof, R. 2012a. Boesenbergia rotunda: From ethnomedicine to drug discovery. Evidence-Based Complementary and Alternative Medicine 2012: 473637.

Tan, E.C., Karsani, S.A., Foo, G.T., Wong, S.M., Abdul Rahman, N., Khalid, N., Othman, S. & Yusof, R. 2012b. Proteomic analysis of cell suspension cultures of Boesenbergia rotunda induced by phenylalanine: Identification of proteins involved in flavonoid and phenylpropanoid biosynthesis pathways. Plant Cell Tissue and Organ Culture 111: 219-229.

Tan, S.K., Pippen, R., Yusof, R., Ibrahim, H., Rahman, N. & Khalid, N. 2005. Simple one-medium formulation regeneration of fingerroot (Boesenbergia rotunda (L.) Mansf. Kulturpfl.) via somatic embryogenesis. In Vitro Cellular and Developmental Biology-Plant 41: 757-761.

Tee, C.S., Lee, P.S., Anna, L.P.K. & Mahmood, M. 2010. Optimisation of protoplast isolation protocols using in vitro leaves of Dendrobium crumenatum (pigeon orchid). African Journal of Agricultural Research 5: 2685-2693.

Tewes, A., Glund, K., Walther, R. & Reinbothe, H. 1984. High yield isolation and recovery of protoplast from suspension cultures of tomato (Lycopersicon esculentum). Zeitschrift fur Pflanzenphysiologie 113: 141-150.

Uchimiya, H. & Murashige, T. 1974. Evaluation of parameters in the isolation of viable protoplast from cultured tobacco cells. Plant Physiology 54: 936-944.

Wu, C.H., Dewir, Y.H., Hahn, E.J. & Paek, K.Y. 2006. Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots of Echinacea angustifolia. Journal of Plant Biology 49: 193-199.

Wu, J. & Lin, L. 2002. Elicitor-like effects of low-energy ultrasound on plant (Panax ginseng) cells: Induction of plant defense responses and secondary metabolite production. Applied Microbiology and Biotechnology 59: 51-57.

Yeong, H.Y., Khalid, N. & Phang, S.M. 2008. Protoplast isolation and regeneration from Gracilaria changii (Gracilariales, Rhodophyta). Journal of Applied Phycology 20: 641-651.

Zhang, J.B., Shen, W.T., Yan, P., Li, X.Y. & Zhou, P. 2011. Factors that influence the yield and viability of protoplasts from Carica papaya L. African Journal of Biotechnology 10: 5137-5142.

Zhang, Y.H., Zhong, J.J. & Yu, J.T. 1996. Enhancement of ginseng saponin production in suspension cultures of Panax notoginseng: Manipulation of medium sucrose. Journal of Biotechnology 51: 49-56.

Zhou, X., Wei, Y., Zhu, H., Wang, Z., Lin, J., Lu, L. & Tang, K. 2008. Protoplast formation, regeneration and transformation from the taxol-producing fungus Ozonium sp. African Journal of Biotechnology 7: 2017-2024.

*Pengarang untuk surat-menyurat; email: lani@um.edu.my