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Calla lily chlorotic spot virus (CCSV), a tospovirus infecting Calla lily (Zantedeschia spp.) in Taiwan, with symptoms of chlorotic spots on leaves and stems, was previously reported serologically but distantly related to Watermelon silver mottle virus (WSMoV). To further characterize the virus, DNA fragments corresponding to the S RNA of the virus were amplified from total RNA extracted from CCSV-infected plants of Nicotiana benthamiana, cloned and sequenced. The full-length viral strand of the S RNA was determined as 3,172 nucleotides in length, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA was found identical to those of other tospoviruses, confirming that CCSV belongs to the genus Tospovirus. The N and the NSs proteins of CCSV share low amino acid identities, 20.9 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. The phylogenetic dendrogram of the N protein of CCSV compared with those of other tospoviruses in WSMoV serogroup indicates that CCSV is a distinct tospovirus. Thus, based on the results of molecular characterization of S RNA, we conclude that CCSV is a new tospovirus species belonging to WSMoV serogroup.

In vitro morphogenesis including somatic embryogenesis was studied in five orchids, Cymbidium, Epidendrum, Oncidium, Paphiopedilum and Phalaenopsis. 1) Sections of pseudobulbs, rhizomes and roots of Cymbidium ensifolium var. misericors were induced to formed totipotent calli on a modified MS medium supplemented with 2,4-D and thidiazuron (TDZ). Subculturable calli formed embryoids on a modified MS medium supplemented with BA or TDZ. The embroids developed into rhizomes and, eventually, rhizomes produced normal plantlets. 2) Small transparent tissues formed from surface and cut ends of root, stem, leaf and flower-stalk intenodes of Epidendrum radicans on a modified 1/2-MS basal medium with or without thidiazuron (TDZ) after 1-2 weeks of culture. In light, the transparent tissues enlarged and turned into green organized masses on most of the explants. The organized masses with young protocorm-like bodies (PLBs) proliferated on a hormone-free basal medium, and TDZ promoted the proliferation rate. Normal plantlets converted from PLBs on the same TDZ-containing medium after 1 months of culture. 3) In Oncidium, embryogenic calli were induced in vitro by using root, stem, leaf and flower stalk internode as explants, and healthy plantlets were successfully obtained from the callus cultures. In addition, direct somatic embryogenesis was established by using leaf segments as explants on a modified 1/2strength MS medium. 4) In Paphiopedilum, totipotent callus was induced from seedderived protocorms. The calli were induced to form “globular granules”, and then these granules transformed into PLBs. Eventually, well-developed plantlets were obtained from these callus-derived PLBs. 5) For Phalaenopsis propagation, seedderived protocorms were induced to formed callus. The “proembryo-like structures” were found on the surface of the callus before formation of PLBs, and these PLBs also could convert into well-developed plantlets. INTRODUCTION In vitro morphogenesis and efficient micropagation protocols of several orchids have been studied. These orchids are usually derived from dividing potted plants or seeds. Propagation of this genus, either by seed or by in vitro culture, is still considered difficult. We obtained three kinds of morphogenesis from callus cultures of Cymbidium ensifolium var misericors: rhizomes, shoot buds and granular embryoids. Healthy plantlets were obtained following regeneration via callus-derived rhizomes and granular embryoid-like structures. Similar morphogenesis was also found in Paphiopedilum, Oncidium, and Phalaenopsis. Practical protocols for mass propagation of these orchids have been developed. Mass suspension cultures of these orchids and plant regeneration from these cells are currently being attempted. Plant Regeneration from Callus Culture of Cymbidium ensifolium var. misericors Explants from rhizomes, pseudobulbs and roots formed callus on 1/2 MS medium supplemented with 10 mg/l 2,4-D and 0.1 mg/l TDZ after 2-3 passages of subculture with a 6-months interval. Callus was maintained well in the presence of 3.3 mg/l 2,4-D combined with 0.1 mg/l TDZ. Three distinct morphogenetic events, including rhizome formation, shoot bud formation and production of globular granules were observed on basal medium either without growth regulator supplements, with 1 mg/l TDZ, or with 5 mg/l BA after 1-2 months of culture. About 40.2 % of callus-derived granules developed 115 Proc. IInd IS on Biotech. of Trop & Subtrop. Species Eds: W.-C. Chang and R. Drew Acta Hort 692, ISHS 2005 into rhizomes when transferred onto a modified 1/10 MS medium for 50 days. Eventually, plantlets were obtained from the shoot buds of the callus-derived rhizomes (Chang and Chang, 1998). Effect of TDZ on Bud Development of Cymbidium sinense Wild Seed-derived rhizomes were able to initiate shoot buds in the presence of 0.01-1 mg/l TDZ. However, the further proliferation of rhizomes was retarded by TDZ. Plantlets (about 10 cm in height) were obtained from rhizome-derived shoot buds on medium supplemented with 0.5 mg/l NAA and 1 g/l after 4 months of culture. About 9.1 % and 73% of regenerated plants flowered normally in the 2 year and the 3 year, respectively. This result indicated that the treatment of TDZ during bud development may shorten the juvenile phase of this orchid species (Chang and Chang, 2000a). In Vitro Flowering of Cymbidium ensifolium var. misericors Callus-derived rhizomes produced flowers precociously on a modified 1/2 MS medium containing NAA with 2iP, BA and TDZ after 100 days of culture. TDZ at 3.3-10 μM or 2iP at 10-33 μM combined with 1.5 μM NAA gave the best response on flower induction. Although the in vitro flowers were smaller in size, their morphology is normal. These flowers bloomed for about two weeks in vitro. The pollens of these in vitro flowers are viable, and germinated on an agar medium after 12 hours of culture (Chang and Chang, 2000b). Efficient Production of Protocorm-like Bodies of Epidendrum Flower stalk internodes formed a large number of PLBs on 1/2 MS medium supplemented of TDZ. These PLBs grew well and proliferated more on the same medium. The best response on PLB production was found at a modified 1/2 MS medium containing 825 mg/l NH4NO3, 950 mg/l KNO3 and 40 g/l sucrose. BA, kinetin and zeatinriboside promoted PLB proliferation and subsequent shoot formation. The optimized procedure required about 12-13 weeks from the initiation of PLBs to plantlet formation. Somatic Embryogenesis and Plant Regeneration of Oncidium Protocols for in vitro plant regeneration via direct or indirect somatic embryogenesis of Oncidium spp. have been established. 1) Leaf segments of the cultivar ‘Gower Ramsey’ were induced to directly form somatic embryos on 1/2-MS medium supplemented with TDZ. Plantlet formation from these somatic embryos was achieved on the same basal medium devoid of plant growth regulators (Chen et al., 1999). 2) Yellowish embryogenic calluses derived from leaf, root and stem explants of the cultivar ‘Gower Ramsey’ were induced and maintained well on 1/2-MS medium supplemented with 2,4-D and TDZ. Somatic embryos formed from these calluses on the same basal medium supplemented with NAA and TDZ. Plantlet conversion was made on the same basal medium devoid of plant growth regulators (Chen and Chang, 2000b; Wu et al., 2004). 3) Flower stalk explants of the cultivar ‘Sweet Sugar’ formed somatic embryos on TDZ-containing medium, and plantlets derived form these embryos were obtained on the same medium (Chen and Chang, 2000a). Leaf segments taken from regenerated plantlets of cv. Sweet Sugar were induced to obtain repetitive somatic embryogenesis (Su et al., in press). The leaf culture system has been further used to study the effects of tissue culture conditions (Chen and Chang, 2002), explant characteristics (Chen and Chang, 2002), plant growth regulators (auxins, auxin inhibitors, cytokinins, GA3, growth retardants, ACC and ethylene inhibitors) (Chen and Chang, 2001; Chang and Chang, 2003) on direct somatic embryogenesis. Plant Regeneration from Protocorm-derived Callus of Paphiopedilum Explants from seed-derived protocorms were induced to form yellowish callus on a 1/2 MS medium supplemented with 1-10 mg/l 2,4-D and 0.1-1 mg/l TDZ. The callus