Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.     

Antioxidants and improved regrowth procedure facilitated cryoconservation of Paphiopedilum insigne Wall. Ex. Lindl. - An Endangered Slipper Orchid

Orchids and their sustainability are very important issues that need global conservation efforts. Paphiopedilum insigne (an endangered orchid), is one of the most excessively exploited species of orchids and is mentioned in the IUCN Red List and Appendix I of CITES. The prospect for conservation and commercialization of this species would be strengthened with the development of improved cryopreservation techniques. This study reports on successful cryopreservation of protocorms of P.insigne after cryopreservation using vitrification (Vit) and encapsulation-vitrification (E-Vit) techniques. The study compared the addition of four antioxidants to the pretreatment and recovery stages, three growth media, and agitated vs. semisolid culture medium for initial recovery. Recovery after cryopreservation for the control was 27% for Vit and 37% for E-Vit. In both cases agitated culture produced improved recovery by about 10%, but with significantly better recovery with E-Vit. The best recovery (51.2 ± 0.9%) was recorded for 0.5 M sucrose precultured encapsulated protocorms treated for 45 min with PVS2 and recovered in ½ MS (L/S) liquid medium for 10 days under agitation, followed by transfer to semi-solid medium. This recovery was further enhanced (62.7 ± 0.5%) with the incorporation of 30 μM glutathione in both liquid preculture and the liquid and semisolid regrowth medium. This new protocol improved the E-Vit cryopreservation recovery from the initial 37%–63%, providing a suitable technique for storage of this threatened orchid.     

In vitro propagation of temperate Australian terrestrial orchids: revisiting asymbiotic compared with symbiotic germination

Using a common temperate herbaceous terrestrial orchid from Australia (Caladenia latifolia) this study investigated 19 asymbiotic media variations comprising four commonly used basal media [half‐strength Murashige and Skoog (½MS), Knudson C (KC), Pa5, and Vacin and Went (VW), with combinations of the plant growth regulators 6‐benzylaminopurine (BA) and α‐naphthalene acetic acid (NAA) or coconut water (CW) and compared their performance with germination on a standard symbiotic germination medium, oatmeal agar (OMA). Percentage germination of seeds every 2 weeks for a total of 8 weeks (five replicates per treatment), time to germination, and growth and development phases in seedlings were recorded. ½MS with 5% (v/v) fresh CW delivered 93% germination, with seedling vigour and development indistinguishable from OMA (95% germination). The same protocol was applied to a further ten species (including the endangered Caladenia huegelii), demonstrating high asymbiotic germination performance (60–93%) across a wide phylogenetic range of terrestrial orchid species. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 556–566.     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

Germination responses of four native terrestrial orchids from south-west Western Australia to temperature and light treatments

We report an investigation into the impact of temperature and illumination on in vitro symbiotic and asymbiotic germination of the threatened taxon Caladenia huegelii, and three other orchid spp. namely—Caladenia latifolia, Microtis media and Pterostylis sanguinea, all species from south-west Western Australia, a recognized biodiversity hotspot. High symbiotic germination on oatmeal agar (OMA + fungal symbionts specific to each species) was recorded in three species in continuous dark incubation i.e. C. huegelii seeds (98 % germination at 25 °C), and M. media and P. sanguinea (93 and 98 % respectively at 20 °C). Highest symbiotic germination for C. latifolia (100 %) was observed at 15 and 20 °C under light treatment (12/12 h light/dark). Low temperature incubation (10 °C) significantly suppressed symbiotic germination/development of seedlings across all species. Asymbiotic media treatments assessed (OMA minus fungal symbionts, Pa₅ and ½ MS), failed to stimulate any germination with C. latifolia seeds at 20 °C in either light or dark treatments after an 8 week incubation period. Seeds of M. media sown onto ½ MS medium resulted in higher germination in all developmental stages (3–5) in dark treatment than OMA and Pa₅. Seeds of P. sanguinea sown onto ½ MS medium resulted in higher overall germination in all developmental stages (3–5) in light and dark incubation compared to OMA and Pa₅. OMA supported the highest asymbiotic germination (100 %) in both light and dark incubation with M. media (only to stage 3) but did not support germination and development with other spp. tested. Caladenia huegelii seeds reached developmental Stage 3 (i.e. germinated), but only on Pa₅ medium and only at a relatively low rate in either light (2.6 %) or dark (2.1 %). Germination was higher and development of seedlings faster overall in all test species in symbiotic compared with asymbiotic media treatments. P. sanguinea seeds demonstrated the best response (among species tested) to asymbiotic germination on ½ MS with 40–53 % of germinated seeds spread over developmental stages 3–5 in light or dark incubation (at 20 °C) respectively. Illumination had no effect on fungal symbiont growth across all species, however incubation temperature treatments (10, 15, 20 and 25 °C) affected fungal growth rate. Growth of the fungal symbionts of C. huegelii, M. media and C. latifolia demonstrated significantly lower activity at 10 °C, but the cumulative radial growth rate of the P. sanguinea fungal symbiont reached 64 cm² after only 2 weeks at all temperatures tested, including 10 °C. The study highlights differences in symbiotic and aysmbiotic germination and early protocorm development in vitro between co-occurring herbaceous terrestrial Australian orchid taxa in response to variations in basal media, temperature and light.     

La fioritura di „Tulipa silvestris“ L. in rapporto alla nutrizione azotata

Many members of the Orchidaceae, the largest vascular plant family in Ecuador, are at risk of extinction. It was therefore considered important to establish an efficient way of clonal propagation based on somatic embryogenesis of Cattleya maxima, a native Ecuadorian orchid. To this end, we evaluated the effect on somatic embryo induction of 12 combinations of 2,4-dichlorophenoxyacetic acid and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea, as well as three kinds of stresses. Protocorms produced 42% of embryogenic calli on 1/2 Murashige and Skoog (1/2 MS) medium, compared to 96.3% when protocorms were stressed for 6 h with 0.3 M NaCl, followed by cultivation on 1/2 MS medium supplemented with 0.1 mg L^? 1 2,4-D. Our data demonstrated that the combination of either salt (0.3 M NaCl) or osmotic stress (0.4 M sorbitol) with subculture on 2,4-D (0.1 mg L^–1) medium significantly increases the percentage of protocorms with embryogenic callus. The number of embryos per embryogenic callus was not significantly different from that obtained after subculture in growth factor-free medium.     

Recovery of transgenic orchid plants with hygromycin selection by particle bombardment to protocorms

Protocorms of orchid (Dendrobium hybrid) were transformed by microprojectile bombardment with a helium-pressured PDS 1000 particle gun. Gold particles coated with plasmid DNA containing ß-glucuronidase (GUS) and hygromycin phosphotransferase (Hpt) marker genes were used. Potentially transformed tissues were identified by active growth on MS medium supplemented with 50mg l^-1 hygromycin. After 4–6 months of continuous selection, 15 hygromycin-resistant lines were recovered. Integration of transgenes into the genome of the transformed protocorms and plantlets were confirmed by GUS histochemical assay and Southern blot hybridization. The transgenic protocorms have gone through propagation for more than 8 months and maintained their transgenic characters. These results indicate that we have established a system for orchid transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants.     

In vitro propagation of Dendrobium aphyllum (Orchidaceae)—seed germination to flowering

This communication describes asymbiotic seed germination, protocorm development, micropropagation and flowering in in vitro and hardened seedlings of Dendrobium aphyllum (Roxb.) C.E.C. Fischer. Effects of four culture media viz., Murashige and Skoog (MS); Phytamax (Sigma Chemical Co. USA; PM); Mitra et al. (M) and Knudson ‘C’ (KC), 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D), peptone and activated charcoal were studied on seed germination and protocorm development. Maximum germination (97 %) was recorded in PM basal medium. Peptone (2.0 gl^?1) remarkably enhanced germination percentage (100 %), vigorous growth, high survival and subsequent development of protocorms, while in activated charcoal the response was not encouraging. BAP improved germination percentage, however, 2,4-D showed noticeably low seed germination. The morphogenetic response of protocorms and nodal segments of in vitro raised seedlings varied depending on type of explants and concentrations and combinations of plant growth regulators used. Stout root system was induced in 1/2PM + 0.5 mgl^?1 IAA. Approximately 10 % of the in vitro raised plants (4–5 cm) with 3–4 leaves flowered in vitro irrespective of flowering season. The well-rooted plants showed 80 % survival under green house conditions and flowering was noticed after 5–6 months in 10 % of hardened plants.     

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l^–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.     

In vitro propagation and field establishment of Eulophia cullenii (Wight) Bl., a critically endangered orchid of Western Ghats, India through culture of seeds and axenic seedling-derived rhizomes

An efficient protocol has been devised for the propagation and field establishment of Eulophia cullenii (Wight) Bl., a terrestrial orchid having ornamental potentialities, and is critically endangered in Western Ghats, India. Seeds extracted from 60–90-d-old capsules germinated in ½ MS, ¼ MS, Knudson C, or Mitra liquid medium developed into 1.4–2.5-mm-diameter protocorms in 60 d. Supplementation of organic additives like coconut water, peptone, yeast extract, and casein acid hydrolysate (CH) significantly enhanced protocorm growth. Upon subculture onto agar-gelled Mitra medium fortified with 0.05% CH, 56% of protocorms regenerated into shoots through the formation of linear mini-rhizomes. The regenerated shoots grew vigorously in ½ MS, producing new rhizomes. Mature rhizomes from axenic seedlings produced maximum (13?±?1.4) shoots/whole rhizome in ½ MS fortified with 44.4 μM 6-benzylaminopurine (BAP), in 120–150 d. Horizontal and longitudinal halves of the rhizome also gave multiple shoots (6–8.5) in the presence of 44.4 μM BAP. Shoots or shoot clumps sub-cultured onto ½ MS basal medium produced roots followed by rhizomes in 60–150 d. Seedlings with mature rhizomes showed 70% establishment in the nursery and added a new rhizome at the end of one growth cycle. An average of 70.6% of the rhizomes originating from seedlings during the second growth cycle sprouted to produce new shoots, when planted in the native localities. Asymbiotic germination and cloning through rhizomes thus can provide a large number of vigorous plants of E. cullenii for ornamental exploitation as well as eco-restoration, if rhizome as storage organ is ensured in the propagule.     

Factors influencing shoot multiplication of lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis>)

Effect of plant growth regulators, explant size, season of explant collection, temperature (20, 25 and 30 °C) and photoperiod on in vitro lotus (Nelumbo nucifera Gaertn.) shoot formation and growth were examined. Shoots formation was greatly influenced by growth regulators, explant size and season of explant collection. The maximum number of shoots were induced from bud explants on Murashige and Skoog (MS) medium containing 4.44 μM benzyladenine (BA) + 0.54 μM α-naphthalene acetic acid (NAA). Explants formed by bud of one expanded and one unexpanded leaf, which was collected in spring gave encouraging results of shoot production. Higher temperature favoured shoot induction and subsequent growth was much better at 25 °C compared to that at 20 and 30 °C.     

Somatic embryogenesis,acclimatization and genetic homogeneity assessment of regenerated plantlets of <Emphasis Type="Italic">Anoectochilus elatus</Emphasis> Lindl., an endangered terrestrial jewel orchid

An efficient in vitro plant regeneration system was established through somatic embryogenesis for Anoectochilus elatus Lindley, an endangered jewel orchid. Direct somatic embryogenesis was achieved from nodal explants (17.4 embryos per explant with 63.4% response) on Mitra medium supplemented with Morel vitamins, thidiazuron (4.54 µM) and ∞-naphthaleneacetic acid (2.69 µM). Simultaneously, a protocol was developed for indirect somatic embryogenesis from internodal explant, produced embryogenic calli and embryos (31.3 embryos with 76.4% response) on same medium amended with 50 mg/L peptone and 5% coconut water. Both types of embryogenic pathways, produced morphologically similar globular embryos in the form of protocorm like bodies and successfully germinated on hormone free Mitra medium supplemented with Morel vitamins. Morpho-histological investigation of the embryo revealed the initiation and developmental features of somatic embryos. In vitro regenerated plantlets were successfully established from heterotrophic to a photoautotrophic stage by reducing the nutrient content in culture media, adjusting temperature and humidity through three step method. During the process, no morphological and physiological abnormalities were observed. Hardened plantlets were successfully acclimatized at poly tunnel chamber with 95% of survival rate. Further, inter simple sequence repeats (ISSRs) molecular markers were used to analyse the genetic homogeneity of regenerated plants. Analysis with this method showed that the homogeneity is comparatively higher in direct somatic embryo regenerated plants (94.22%) as compared to plants elevated from an indirect somatic embryo (93.05%). The present study provides morpho-histological and genetically stable plants for germplasm conservation and further utility of this endangered jewel orchid.     

The Effect of Seed Cryopreservation on the Development of Protocorms by the Hybrid Orchid Bratonia

The aim of this work was to study the influence of storage in liquid nitrogen on the viability of seeds of the hybrid orchid Bratonia and further development of its protocorms in vitro. Seeds were frozen in ampoules by direct immersion in liquid nitrogen and stored in the cryobank for a month. The germination rates of cryopreserved and control (nonfrozen) seeds did not differ and remained as high as 100%. The protocorms derived were cultured on the agar-solidified Murashige and Skoog nutrient medium (MS), half-strength MS and Knop media and also in Morel liquid medium. During the first 45 days of culturing, protocorms derived from cryopreserved seeds grew faster than control protocorms on the MS and half-strength MS media but, at longer culturing (496 days), the size of control protocorms was significantly larger. After 639 days of culturing, there was no difference in the amount of perished, budding, and newly formed protocorms obtained from cryopreserved and control seeds, except half-strength MS medium where the number of budding protocorms in the case of cryopreserved seeds was a little greater than in the control treatment. After seed cryopreservation, the frequency of budding and newly formed protocorms was greater on the agarized MS and in liquid Morel media. Cryopreservation had little effect on the subsequent growth of protocorms in vitro. The preferable nutrient media for culturing the protocorms have been suggested.     

COMPARATIVE STUDY ON THE PHOTOSYNTHETIC PROPERTIES OF PRASIOLA (CHLOROPHYCEAE) AND NOSTOC (CYANOPHYCEAE) FROM ANTARCTIC AND NON‐ANTARCTIC SITES1

The terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and on the Antarctic continent. The terrestrial green alga Prasiola crispa (Lightf.) Kütz. is also distributed in Antarctica. These two species need to acclimate to the severe Antarctic climate including low ambient temperature and desiccation under strong light conditions. To clarify this acclimation process, the physiological characteristics of the photosynthetic systems of these two Antarctic terrestrial organisms were assessed. The relative rate of photosynthetic electron flow in N. commune collected in Japan and in Antarctica reached maxima at 900 and 1,100 μmol photons · m^?2 · s^?1, respectively. The difference seemed to reflect the presence of high amounts of UV‐absorbing substances within the Antarctic cyanobacterium. On the other hand, the optimal temperatures for photosynthesis at the two locations were 30°C–35°C and 20°C–25°C, respectively. This finding suggested a decreased photosynthetic thermotolerance in the Antarctic strain. P. crispa exhibited desiccation tolerance and dehydration‐induced quenching of PSII fluorescence. Re‐reduction of the photooxidized PSI reaction center, P700, was also inhibited at fully dry states. Photosynthetic electron flow in P. crispa reached a maximum at 20°C–25°C and at a light intensity of 700 μmol photons ? m^?2 ? s^?1. Interestingly, the osmolarity of P. crispa cells suggested that photosynthesis is performed using water absorbed in a liquid form rather than water absorbed from the air. Overall, these data suggest that these two species have acclimated to optimally photosynthesize under conditions of the highest light intensity and the highest temperature for their habitat in Antarctica.     

Comparative longevity of Australian orchid (Orchidaceae) seeds under experimental and low temperature storage conditions

Seeds from ten terrestrial orchid species, nine from the south‐west Australian biodiversity hotspot (Caladenia arenicola, Caladenia flava, Caladenia huegelii, Diuris laxiflora, Microtis media ssp. media, Pterostylis recurva, Pterostylis sanguinea, Thelymitra crinita and Thelymitra macrophylla) and one from south‐east Australia (Diuris fragrantissima), were placed into experimental storage to assess their relative longevity and likely optimal conditions for long‐term conservation seed banking. Seeds from all species were desiccation tolerant, germinating after drying at 23% relative humidity (C. arenicola, C. huegelii, P. sanguinea and T. macrophylla) or 5% relative humidity (C. flava, D. laxiflora, M. media ssp. media, P. recurva and T. crinita) at 23 °C. From automatedly determined moisture adsorption and desorption isotherms at 23 °C, these equate to tolerance of drying to 0.03–0.06 g water g^?1 dry weight or 0.013–0.028 g water g^?1 dry weight, respectively. Results of storage experiments at a range of moisture contents and temperatures suggest conventional seed bank storage at ?18 °C after equilibration at c. 23% relative humidity (at 23 °C) may be suitable for most of the species, although there was higher germination of P. recurva seeds stored at ?80 °C and of M. media ssp. media seeds equilibrated at 75% relative humidity. However, there was considerable variation in germination of seeds sampled after different storage periods, making it difficult to identify optimal storage conditions definitively. Results of comparative longevity storage experiments at 60% relative humidity and 40 °C suggest seeds from these orchid species are short‐lived compared with non‐orchid species, and with Australian species in particular. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 26–41.     

High efficiency of stable genetic transformation in <Emphasis Type="Italic">Dendrobium via</Emphasis> microprojectile bombardment

Several parameters affecting transient β-glucuronidase (GUS) expression in protocorms of Dendrobium cv. Jaquelyn Thomas were examined using bombardment technique with pActin-1D plasmid. The GUS activity in Dendrobium protocorm was not significantly affected by size of the target, type of particles, and helium gas pressure. However, the numbers of surviving tissues after bombardment were different. Transgenic orchids were established by bombardment of actively dividing protocorms with gold particles coated with pMNK1005 plasmid containing hygromycin phosphotransferase (hgh) and green fluorescent protein (gfp) genes driven by a ubiquitin promoter. A high efficiency of orchid transformation was established using three selection steps. The bombarded protocorms were screened on medium supplemented with 5 mg dm⁻³ and 25 mg dm⁻³ hygromycin and surviving protocorms were stringently selected on medium containing 30 mg dm⁻³ hygromycin. The transformation efficiency was 19.87 % and GFP expressing protocorms were not chimeras. The integration of the transgene into genomic DNA of transgenic plantlets was confirmed by PCR and Southern blot hybridization. All but one of the transgenic lines contained multiple copies of the transgene.     

墨兰原球茎生长的研究

以墨兰种子胚经培养诱导萌发形成的原球茎为材料,研究其生长的最适条件。结果表明：最佳基本培养基是ＭＳ培养基;ＮＡＡ０．５ｍｇＬ＋ＢＡ１．０ｍｇＬ－１有利于生长和分化;在培养基中加入１％蔗糖和０．１％活性炭最有利于原球茎生长;ｐＨ５．０偏酸环境适合于生长;每天给予１５．５ｕｍｏｌｍ－２Ｓ－１１６ｈ的光照最佳。如将上述单因子组合在一起,其原球茎鲜重增长率比对照（ＭＳ培养基）高４６％。在不同切割方式中,掰开法鲜重增长率比纵切法或横切法都高。     

Asymbiotic seed germination,in vitro seedling development,and greenhouse acclimatization of the threatened terrestrial orchid <Emphasis Type="Italic">Bletia purpurea</Emphasis>

Procedures for asymbiotic seed germination and seedling acclimatization were developed for Bletia purpurea, a threatened North America native terrestrial orchid. Six asymbiotic orchid seed germination media (Knudson C, PhytoTechnology Orchid Seed Sowing Medium, Malmgren Modified Terrestrial Orchid Medium, Vacin &; Went Modified Orchid Medium, ½-strengh Murashige &; Skoog, and BM-1 Terrestrial Orchid Medium) were examined for their effectiveness in promoting seed germination and protocorm development of B. purpurea in either a 0/24 h or 16/8 h L/D photoperiod. Germination occurred regardless of medium or photoperiod treatment. However, advanced seedling development (Stage 6) only occurred on Vacin &; Went Modified Orchid Medium in the 16/8 h L/D photoperiod. Further effects of photoperiod on in vitro seedling development were also examined. Shoot length, leaf width, root number and length, and fresh weight and dry weight in the 16/8 h L/D photoperiod were all significantly different when compared to the 8/16 h and 12/12 L/D photoperiods. In vitro seedlings were readily acclimatized to greenhouse conditions. Seedlings showed high survival all potting media. Seedlings acclimatized in Fafard Mix 4 potting medium developed significantly longer roots. Corm formation occurred regardless of potting media used.     

Germination of mature seeds of <Emphasis Type="Italic">Calanthe tricarinata</Emphasis> Lindl., an endangered terrestrial orchid,by asymbiotic culture <Emphasis Type="Italic">in vitro</Emphasis>

The effects of culture conditions on the asymbiotic germination of mature seeds of Calanthe tricarinata Lindl., an endangered terrestrial cool-climate orchid, were examined. Specifically, conditions such as illumination, temperature, and the addition of plant growth regulators to the medium were studied. Mature seeds were harvested from plants that had been collected in Toyama Prefecture, Japan, and maintained at the Botanic Gardens of Toyama. Solidified “New Dogashima” medium was used as the basal medium, and it was supplemented with 6-benzyladenopurine (BA) or α-naphthalene acetic acid (NAA). White light at 40 μmol m⁻² s⁻¹, with a 16-h photoperiod, inhibited the germination of seeds by 53–80%, as compared to dark controls in genotypes examined. The optimal temperature for the germination of seeds in darkness was 20°C and the germination frequency reached 60%, whereas it was only 28% at 25°C. While both NAA and BA stimulated germination, BA was more effective than NAA. After storage for 18 mo at 5°C, seeds incubated on medium that contained 0.2 mg l⁻¹ BA germinated at a frequency of 36%, which was twice that of seeds grown without any plant growth regulators. The frequency of subsequent germination decreased during storage of seeds at 5°C for approximately 2 yr, dropping from 61% to 13%. The protocorms obtained in this study were developed to plantlets readily after transferring to fresh 1/2 MS medium without any plant growth regulators. They were successfully acclimatized in green house after two to three subcultures in vitro. The significant role of a reproducible protocol for the germination of mature seeds is discussed in terms of the ex situ conservation of endangered orchid species.     

Drying rate and dehydrin synthesis associated with abscisic acid-induced dehydration tolerance in Spathoglottis plicata orchidaceae protocorms

Dehydration tolerance of in vitro orchid protocorms was investigated under controlled drying conditions and after abscisic acid (ABA) pretreatment. Protocorms were obtained by germinating seeds on Murashige and Skoog (MS) medium containing 10% (v/v) coconut water, 2% (w/v) sucrose and 0.8% (w/v) agar, and were dehydrated in relative humidities (RH) ranging from 7% to 93% at 25 degrees C. The critical water content of dehydration tolerance was determined, using the electrolyte leakage method. Drying rate affected the critical water content. Slow drying under high RH conditions achieved the greatest tolerance to dehydration. ABA pretreatment decreased the drying rate of protocorms, and increased dehydration tolerance. Improved tolerance to dehydration after ABA treatment was correlated with the effect of ABA on drying rate of protocorms. When critical water content of protocorms dried under different RH was plotted as a function of actual drying rate, no significant difference in tolerance to dehydration was observed between ABA-treated and control protocorms. ABA pretreatment and dehydration of orchid protocorms induced the synthesis of dehydrin, especially under the slow drying conditions. ABA pretreatment also promoted dry matter accumulation such as carbohydrates and soluble proteins and increased the concentration of K(+) and Na(+) ions in protocorms. The ABA-induced decrease in drying rate was correlated with lower osmotic potential, the enhanced maturity of protocorms and the accumulation of dehydrin in protocorms during pretreatment.