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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 KHALID1,2*
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
Received: 26 September 2014/Accepted: 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 REFERENCES
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.
*Corresponding author; email: lani@um.edu.my
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