April - June 2007 - Kasetsart University

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Kasetsart J. (Nat. Sci.) 41 : 300 - 310 (2007) Protoplast Isolation and Culture of Aquatic Plant Cryptocoryne wendtii De Wit Kanchanaree Pongchawee 1 *, Uthairat Na-Nakorn 2 , Siranut Lamseejan 3 , Supawadee Poompuang 2 and Salak Phansiri 4 ABSTRACT The optimum conditions for protoplast isolation and culture of Cryptocoryne wendtii De Wit were investigated. Protoplasts were successfully isolated from in vitro four-week-old leaves using an enzyme mixture comprising 2% Cellulase Onozuka R-10, 0.2% Pectolyase Y-23, 0.5 M mannitol, 2.5 mM CaCl 2.2H 2O and 5 mM 2 (N-morpholino)-ethanesulfonic acid (MES), pH 5.6. Approximately 1.04±0.06 × 10 7 protoplasts per gram fresh weight with 90.79±4.80% viability were obtained after incubating in enzyme solution for 4 hours in the dark and purified with 16 % sucrose gradient centrifugation. Protoplasts were cultured on modified MS medium supplemented with 0.2 mg/l 2,4dicholorophenoxyacetic acid (2,4-D), 1 mg/l α-naphthalene acetic acid (NAA), 0.5 mg/l zeatin, 0.15 M sucrose and 0.3 M mannitol by agarose-bead with thin layer liquid culture. The protoplasts regenerated cell walls within 24 hours. First cell division was observed after culturing for 2-3 days, and microcolonies were formed within 4 weeks. Enzyme mixture, osmotic solution, incubation time, age of leaves, and sucrose solution concentration were found to influence both yield and viability of protoplasts. Culture media, plant growth regulators and method of culture affected protoplast division. Key words: aquatic plant, Cryptocoryne wendtii De Wit, protoplasts isolation, protoplasts culture INTRODUCTION The Cryptocoryne genus is a member of Araceae with more than 50 different species. They are distributed throughout Southeast Asian coastal zones. Some species are commercially cultivated as aquarium plants (Mühlberg, 1982). Cryptocoryne wendtii De Wit is an important species used in the aquarium plant trade (Rajaj and Horeman, 1977). It is a medium-sized species with thin rhizomes and runners, able to grow 1 Aquatic plant and Ornamental Fish Research Institute, Bangkok 10900 , Thailand. 2 Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand. 3 Gamma Irradiation Service and Nuclear Technology Research Center, Kasetsart University, Bangkok 10900, Thailand. 4 Scientific Equipment Center, KURDI, Kasetsart University, Bangkok 10900, Thailand. * Corresponding author, e-mail: kanchanp@fisheries.go.th emerged or submersed and is propagated by runners (Mühlberg, 1982). The aerial leaves are oblong with round or heart shaped base, 8 to 10 cm long by 2 to 3 cm wide and below water. The blade are narrower (Allgayer and Teton, 1986). In order to increase the value of exports and to cope with international market demand, the improvement of new aquatic plant varieties for desirable traits such as variable leaf color and form are the key to success. Related or relevant genera of cultivated crops contain a large reservoir of Received date : 19/09/06 Accepted date : 22/01/07
genes covering a variety of desirable traits (Liu et al., 2005). However, reproductive incompatibility generally prevents simple hybridization between taxa. Somatic cell fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers (Davey et al., 2005). There have been many reports of the transfer of useful agronomic traits by protoplast fusion for production of triploid (Fu et al., 2003) and polyploid (Mizuhiro et al., 2001) plants and increasing plant vigour (Cheng et al., 2003). This technique may be a possible alternative for the genetic improvement of Cryptocoryne. For successful protoplast fusion, a reliable procedure for protoplast isolation and culture is a prerequisite. Up to date, there are a few reports of protoplast isolation and culture of aquatic plants such as seagrass (Balestri and Cinelli, 2001). In this study, the procedures for isolation and culture of C. wendtii protoplasts were established for the first time. The information obtained from this study will greatly benefit further genetic improvement of Cryptocoryne. MATERIALS AND METHODS Plant materials Shoot tip explants of C. wendtii were surface-sterilized by immersion in 50% (V/V) Kasetsart J. (Nat. Sci.) 41(2) 301 ethanol for 1 min and 1.05 % NaOCl containing 1 drop of Tween-20 per 100 ml for 12 min, followed by rinsing three times with sterile distilled water (Kane et al., 1999). Explants were cultured on semi-solid MS medium (Murashige and Skoog, 1962) supplemented with 2 mg/l 6-benzyladenine (BA), 0.25 mg/l NAA, 30 g/l sucrose and 1.6 g/l gelrite (Sigma, USA). The cultures were incubated under 16/8 h light/dark photoperiod at 25°C. Plantlets derived from shoot tips were subcultured into the same medium every four weeks. Leaves of plantlets were used as the explants for protoplast isolation. Factors affecting the protoplast isolation 1. Enzyme mixtures Five enzyme mixtures (Table 1) were examined for the suitable protoplast isolation. The tested enzyme mixtures were dissolved in 0.5 M mannitol, 2.5 mM CaCl 2.2H 2O and 5 mM 2-Nmorpholino-ethanesulfonic acid (MES) pH 5.6. One gram of in vitro four-week-old leaves were cut transversely into 1-2 mm wide strips in a washing solution (0.45 M mannitol, 2.5 mM CaCl 2.2H 2O and 5 mM MES, pH 5.6). The sliced tissues were plasmolysed by immersion in washing solution for 30 minutes. The plasmolysis solution was pipetted off, replaced with 5 ml of the filtersterilized (Satorius, pore size 0.20 µm) enzyme mixtures and incubated in the dark on a gyratory shaker (40 rpm) at 25°C for 4 hr. The protoplasts Table 1 Components of enzyme mixtures used for protoplast isolation of C. wendtii Enzyme Enzyme concentration (% w/v) mixtures Cellulase Pectinase Cellulase R-10a Cellulase RSa Macerozyme R-10a Pectolyase Y-23b E1 2 - 2 - E2 2 - - 0.2 E3 - 2 2 - E4 - 2 - 0.2 E5 2 2 0.1 a Yakult, Tokyo. b Seishin, Tokyo.
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April - June 2007 Volume 41 Number
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KASETSART JOURNAL NATURAL SCIENCE T
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Kasetsart J. (Nat. Sci.) 41 : 205 -
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harvesting time which started from
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y Chen and Paull (2000). Figure 1 a
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pineapple fruit allowed the accumul
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Kasetsart J. (Nat. Sci.) 41(2) 215
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Cluster analysis Cluster analysis u
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Table 3 (Continued) 1 2 3 4 5 6 7 8
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CONCLUSION AFLP markers classified
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difference of soil texture used in
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under wet soil condition planting.
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tropics. Following the expansion, w
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sealed in a plastic box with 1 cm w
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moisture content increment after so
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Frequency Frequency 30 20 10 0 20.0
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School, Kasetsart University. The a
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for combining ability of lines (Lam
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selective mass sibbing within indiv
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Although most of S 2-interfamily hy
Page 49 and 50: composite sets as used in this stud
Page 51 and 52: Kasetsart J. (Nat. Sci.) 41 : 251 -
Page 53 and 54: days. The numbers of calli producin
Page 55 and 56: the report of Ching (1982) showing
Page 57 and 58: clearly amplified bands generated b
Page 59 and 60: caused by either the recombination
Page 61 and 62: Kuhlmann, V. and B. Foroughi-Wehr.
Page 63 and 64: Xanthomonas fuscans subsp. aurantif
Page 65 and 66: Kasetsart J. (Nat. Sci.) 41(2) 265
Page 67 and 68: was collected and washed with 70% e
Page 69 and 70: expected size was amplified with 35
Page 71 and 72: eaction technique has been used for
Page 73 and 74: Schaad, N.W., D. Opgenorth and P. G
Page 75 and 76: MATERIALS AND METHODS Model descrip
Page 79 and 80: calculated TF value calculated TF v
Page 81 and 82: sincere gratitude and deep apprecia
Page 83 and 84: 1989). In monogastrics, the inclusi
Page 85 and 86: of all LLS samples in this study we
Page 87 and 88: Melbourne, Parkville, Victoria. Goe
Page 89 and 90: considered responsible for low prod
Page 93 and 94: mineral supplementation suggested b
Page 95 and 96: Body weight chane (kg/head) 32 30 2
Page 97 and 98: CONCLUSION AND RECOMMENDATION With
Page 99: SAS. (Statistical Analysis System).
Page 103 and 104: mg/l NAA and 0.5 mg/l zeatin) incub
Page 105 and 106: 5. Purification by various sucrose
Page 107 and 108: as the culture period increased. So
Page 109 and 110: culture, pp. 1-20. In L.C. Fowke an
Page 113 and 114: GC. Analytical methods Total carboh
Page 115 and 116: ash. The crude hot water polysaccha
Page 117 and 118: spirulan (H-SP), and a desulfated c
Page 121 and 122: cells often displayed an increased
Page 123 and 124: LITERATURE CITED Bell, T.A. and D.V
Page 125 and 126: for alternative sources of supply.
Page 127 and 128: BR2.1.2 formed the same clade with
Page 129 and 130: supplement with glutamate (Iida et
Page 131 and 132: optimal for S. limacinum BR2.1.2 fo
Page 133 and 134: fluorescent lamp at 1.5 kLux develo
Page 137 and 138: Wisconsin, USA). Total extracted RN
Page 139 and 140: RESULTS Cloning and sequencing of m
Page 141 and 142: Expression of rmIL-2 and purificati
Page 143 and 144: other strains of mice have differen
Page 145 and 146: dose interleukin-2 therapy promotes
Page 147 and 148: The chitosano-oligosaccharides are
Page 149 and 150: enzyme and cells were maximized by
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of crude chitosanases. Fortunately,
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Relative activity (%) 100 80 60 40
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Uchida, K. Tateishi, O. Shida and K
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MATERIALS AND METHODS 1. Materials
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without coating and coated with pec
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in Table 5-7. It was found that the
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the cultures at 10,200 × g for 15
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incubation and reached the final pH
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y lysine- and tyrosine-decarboxylas
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during the stages of ripening as su
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As the 25 companies were divided in
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All the data comes up with informat
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as piece “A” or straight as pie
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et al. (1998); Zeng (2000); and Tal
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p 1, i * Kasetsart J. (Nat. Sci.) 4
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c ≤ c2 jq2 j n q p k p * 2, j , ,
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algorithm would consider RM 5, prio
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the maximum deviation of about 10%.
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Stock Quantities Using Heuristic Op
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which dynamically and automatically
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2. Framework architecture and compo
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Risk (VaR) calculation which was im
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Throughput (job/sec) 7 6 5 4 3 2 1
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Speed up 40 35 30 25 20 15 10 5 0 F
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and discovery, resource selection a
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April - June 2007 - Kasetsart University

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