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

 

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

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

 

 

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