THAI JOURNAL OF BOTANY 2 (Special Issue): 151-154. 2010.

วารสารพฤกษศาสตรไทย 2 (ฉบับพิเศษ): 151-154. 2553.

Cryopreservation of nigra (Gaertn.) Burtt by Vitrifi cation-based technique

CHUSANA RUNGJINDAMAI1, KANCHIT THAMMASIRI2,*, NGARMNIJ CHUENBOONNGARM2 & THAYA JENJITTIKUL2

1 Master of Science Programme in Science, Faculty of Science and Faculty of Pharmacy, Bangkok 10400, 2 Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

ABSTRACT. In the cryopreservation of Alpinia nigra (Gaertn.) Burtt, shoot tips from in vitro plantlets cultured on Murashige and Skoog (1962) (MS) agar medium for 1 month were dehydrated with a preculture solution (0.3 M sucrose) for 3 days on a rotary shaker at 100 rpm and subsequently exposed to a cryoprotectant solution (5% w/v DMSO and 5% w/v glycerol) and a loading solution (0.4 M sucrose and 2 M glycerol) for 20 min in each solution. They were kept in 1.8 ml cryotubes, containing PVS2 solution [0.4 M sucrose, 15% (w/v) ethylene glycol, 15% (w/v) DMSO and 30% (w/v) glycerol] for 0, 20, 40, 60, 80 or 100 min, respectively, before being immediately plunged into liquid nitrogen for 1 hour. Thereafter, cryotubes were taken out of a liquid nitrogen tank and rapidly warmed in a waterbath at 40˚C for 2 min. PVS2 solution was replaced with an unloading solution (1.2 M sucrose) for 20 min before shoot tips were cultured on MS agar medium supplemented with 1 ppm TDZ for 1 month and subsequently transferred to PGRs free MS agar medium for 1 month. The shoot tips exposed to PVS2 solution for 80 min showed the highest percentage of shoot and development at 27.

KEYWORDS: Cryopreservation, Alpinia nigra, PGRs, TDZ

INTRODUCTION accents, and cut fl owers. They are widely used as medicines (e.g. A. offi cinarum) in Alpinia is the largest and the most , , and some countries in Asia, and widespread genus in , which as and condiment (e.g. A. galanga) consists of about 230 species. They occur (Kress et al., 2005). Alpinia nigra (Gaertn.) from to east and , Burtt is a native species in Nonthaburi Australia, and the Pacifi c islands. Several province. It is not only used as a and a species are important ornamental (e.g. medicinal plant, but also as an edible A. purpurata) as pot plants, landscape vegetable. The young shoots and young

* Corresponding author: [email protected] 152 Chusana Rungjindamai et al. infl orescences were used as the ingredients preculture solution (0.3 M sucrose) for 3 days of several native Thai food (Jenjittikul, on a rotary shaker at 100 rpm and subsequently 2004). exposed to a cryoprotectant solution (5% w/v DMSO and 5% w/v glycerol) and a loading In vitro conservation of genera is solution (0.4 M sucrose and 2 M glycerol) suitable for protecting genetic resources from for 20 min in each solution. They were kept epidemic diseases and other natural disasters in 1.8 ml cryotubes containing PVS2 solution (Yamuna et al., 2007), however, in vitro [0.4 M sucrose, 15% (w/v) ethylene glycol, culture is expensive for manipulation, easy 15% (w/v) DMSO and 30% (w/v) glycerol] for contamination and somaclonal variation for 0, 20, 40, 60, 80 or 100 min at room which increase with time. temperature (~25˚C), respectively, before Cryopreservation using liquid nitrogen being immediately plunged into liquid (-196˚C) is a suitable technique for long-term nitrogen for 0 (treated control) and 1 hour. storage without genetic alteration because Thereafter, cryotubes were taken out of a extreme low temperature stops metabolic liquid nitrogen tank and rapidly warmed in activities (Ray & Bhattacharya, 2008). This a waterbath at 40˚C for 2 min. PVS2 solution method is better than in vitro conservation was replaced with an unloading solution (1.2 because of low cost and easiness to M sucrose) for 20 min before shoot tips were manipulate. The most important problem of cultured on Murashige and Skoog (1962) cryopreservation is the formation of ice (MS) agar medium supplemented with 1 ppm crystal from plant cells, which could destroy TDZ for 1 month and subsequently transferred cell membrane. The successful technique to to PGRs free MS agar medium containing prevent ice crystal formation differs among 30 g/l sucrose and 8 g/l agar (pH 5.8) at room cultures of various plant species (Karl- temperature under 16 h/d photoperiod for 1 Hermann et al., 2009). Vitrifi cation-based month. technique is effectively used in several plant species such as ginger (Zingiber offi cinale) RESULTS AND DISCUSSION (Yamuna et al., 2007), Dioscorea rotundata (Mandal et al., 2008) and white poplar Thidiazuron (TDZ) is a plant growth (Populus alba) (Lambardi et al., 2000). regulator (PGR) used at low concentration Method for cryopreservation of Alpinia nigra for stimulating high percentage of axillary shoot tips was investigated using vitrifi cation shoot proliferation in many woody plant technique. species (Huetteman & Preece, 1993; Sajid & Aftab, 2009) and in Zingiberaceae, such as Curcuma longa (Prathanturarug et al., 2003) METHODOLOGY Gagnepainia spp. (Prathanturarug et al., Shoot tips (1-2 mm long) were excised 2007) and Amomum krervanh (Tefera & under stereo microscope from 2-3 cm long Wannakraijoy, 2004) etc. TDZ is a strong plantlets. They were dehydrated with cytokinin used for enhancing shoot Cryopreservation oݦ Alpinia nigra by Vitri ication-based technique 153

FIGURE 1. Non-cryoperserved (-LN) and cryoperserved (+LN) A. nigra shoot tips exposed to PVS2 solution for 0 min (left plate) and 80 min (right plate) after 2 months of culture on MS agar medium supplemented with 1 ppm TDZ for 1 month and subsequently transferred to PGRs free MS agar medium for 1 month.

100

80

60 + LN - LN (Treated control) 40

20

Percentage of regrowth (%) 0 0 20 40 60 80 100 Exposure time to PVS2 solution (min)

FIGURE 2. Effects of exposure time to PVS2 solution on regrowth percentage of A. nigra shoot tips by vitrifi cation and exposed to PVS2 solution without plunge in LN (treated control). The data were collected after 2 months of culture. Bars represent standard deviation. production in several species. High dose of shown). Thus, 1 ppm TDZ was used in TDZ can reduce shoot elongation and recovery medium after cryopreservation for increase callus formation (Huetteman & stimulating shoot growth in this experiment. Preece, 1993). Micropropagation of A. nigra The highest percentage of regrowth (27%) on MS medium supplemented with 1 ppm from cryopreserved shoot tips, exposed to TDZ resulted in more shoots than that on MS PVS2 solution for 80 min was observed, medium supplemented with BA (data not whereas 87% of the shoot tips survived in 154 Chusana Rungjindamai et al. treated control (-LN) (Fig. 2). No Mandal, B.B., Ahuja-Ghosh, S. & Srivastava, P.S. cryopreserved shoot tips showed regrowth 2008. Cryoperservation of Dioscorea when exposed to PVS2 solution for 0 min rotundata Poir. : A comparative study with but all shoot tips in treated control survived two cryogenic procedures and assessment (Figs. 1 & 2). Shoot tips treated with PVS2 of true-to-type of regenerants by RAPD solution had a decreased survival rate analysis. CryoLetters 29: 399-408. because the chemical toxicity of PVS2 Murashige, T. & Skoog, F. 1962. A revised medium for rapid growth and bioassays solution increased with time (Yamuna et al., with tobacco tissue cultures. Physiologia 2007). The results showed that 80 min Plantarum 15: 473–497. exposure time to PVS2 solution was optimum Prathanturarug, S., Jenjittikul, T., Angsumalee, for A. nigra shoot tips. This is the fi rst report D., Huadsuwan, P., Kiatseesakul, I., on cryopreservation of A. nigra shoot tips by Duangnet, J., Chuakul, W. & Saralamp, P. vitrification method. Further study with 2007. Micropropagation of Gagnepainia different cryopreservation methods should godefroyi K. Schum. and Gagnepainia be carried out. thoreliana (Baill.) K. Schum. – Rare medicinal plant of Thailand. Journal of Biochemistry REFERENCES & Biotechnology 16(2): 135-137. Prathanturarug, S., Soonthornchareonnon, N., Huetteman, C.A. & Preece, J.E. 1993. Thidiazuron: Chuakul, W., Phaidee, Y. & Saralamp, P. a potent cytokinin for woody plant tissue 2003. High-frequency shoot multiplication culture. Plant Cell, Tissue and Organ in Curcuma longa L. using thidiazuron. Culture 33: 105-119. Plant Cell Reports 21: 1054-1059. Jenjittikul, T. 2004. Alpinia nigra. In: Ray, A. & Bhattacharya, S. 2008. Cryopreservation of Plant in Thai letter . pp. 65-66. Royal of in vitro grown nodal segments of Rauvolfa Institute of Thailand, Bangkok. serpentine by PVS2 vitrifi cation. CryoLetters Karl-Hermann, N., Ashwani, K. & Jafargholi, 29: 321-328. I. 2009. Callus cultures: Maintenance of Sajid, Z.A. & Aftab, F. 2009. Effect of thidiazuron Strains, Cryopreservation. In: Plant (TDZ) on in vitro micropropagation of Cell and Tissue Culture - A Tool in Solanum tuberosum L. cvs. Desiree and Biotechnology Basics and Application, pp. Cardinal. Pakistan Journal of Botany 29-31. Springer-Verlag Berlin, Heidelberg. 41:1811-1815. Kress, W.J., Liu, A.Z., Newman, M., & Li, Q.J. Tefera, W. & Wannakraijoy, S. 2004. 2005. The molecular phylogeny of Alpinia Micropropagation of Krawan (Amomum (Zingiberaceae): a complex and polyphyletic krervanh Pierre ex Gagnep.). ScienceAsia genus of . American Journal of 30: 9-15. Botany 92: 167-178. Yamuna, G., Sumathi, V., Geetha, S.P., Praveen, Lambardi, M., Fabbri, A. & Caccavale, A. 2000. K., Swapna, N. & Nirmal, B.K. 2007. Cryopreservation of white poplar (Populus Cryopreservation of in vitro grown shoots alba L.) by vitrifi cation of in vitro-grown of ginger (Zingiber officinale Rosc.). shoot tips. Plant Cell Reports 19: 213-218. CryoLetters 28: 241-252.