Comparative transcriptomic analyses of normal and peloric mutant flowers in Cymbidium goeringii Rchb.f identifies differentially expressed genes associated with floral development.

Cymbidium geringii has high ornamental and economic importance. Its traits, including flower shape, size, and color, are highly sought by orchid breeders. Gaining insights into the molecular basis of C. geringi flower development would accelerate genetic improvement of other orchids. Methods and Results: Here, C. goeringii RNA was purified from normal and peloric mutant flowers, and cDNA libraries constructed for Illumina sequencing. We generated 329,156,782 clean reads, integrated them, and then assembled into 236,811 unigenes averaging 595 bp long. A total of 11,992 differentially expressed genes s, of which 6119 were upregulated and 5873 downregulated, were uncovered in peloric mutant flower buds relative to normal flower buds. Kyoto Encyclopedia of Genes and Genomes enrichment assessments posited that these differentially expressed genes are associated with "Photosynthesis", "Linoleic acid metabolism", as well as "Plant hormone signal transduction" cascades. The DEGs were designated to 12 remarkably enriched GO terms, and 16 cell wall associated GO terms. The expression level of 16 determined genes were verified using RT-qPCR. Conclusions: Our gene expression data may be used to study the regulatory mechanism of flower organ development in C. geringi.

Abscisic Acid Inhibits Asymbiotic Germination of Immature Seeds of Paphiopedilum armeniacum.

Paphiopedilum armeniacum is a rare orchid native to China with high ornamental value. The germination of P. armeniacum seeds is difficult, especially for the mature seeds, which is the major limitation for their large-scale reproduction. This study explored the reasons for seed germination inhibition from the aspects of the important plant endogenous hormone-abscisic acid (ABA). The major endogenous hormone contents of seeds were determined at different developmental stages. The ABA content was 5.8 ng/g in 73 days after pollination (DAP) for the immature seeds, peaked at 14.6 ng/g in 129 DAP seeds, and dropped to 2.6 ng/g in the late mature stage of the 150 DAP seeds. The reduction of ABA content in the mature seed suggests a possible contribution to the increased expression of CYP707A, an ABA catabolism gene. The germination rate of the immature seeds was reduced to 9% from 69% when 5 µg/mL ABA was added to the Hyponex N026 germination medium. The result showed that ABA can inhibit the germination of P. armeniacum immature seeds. However, for the heavily lignified mature seeds, reduction in endogenous ABA level does not result in an increase in the germination rate. Lignin accumulation in the seed coat imposes the physical dormancy for P. armeniacum. In summary, the germination of P. armeniacum is regulated by both ABA and lignin accumulation.

Identification and Characterization of NPR1 and PR1 Homologs in Cymbidium orchids in Response to Multiple Hormones, Salinity and Viral Stresses.

The plant nonexpressor of pathogenesis-related 1 (NPR1) and pathogenesis-associated 1 (PR1) genes play fundamental roles in plant immunity response, as well as abiotic-stress tolerance. Nevertheless, comprehensive identification and characterization of NPR1 and PR1 homologs has not been conducted to date in Cymbidium orchids, a valuable industrial crop cultivated as ornamental and medicinal plants worldwide. Herein, three NPR1-like (referred to as CsNPR1-1, CsNPR1-2, and CsNPR1-3) and two PR1-like (CsPR1-1 and CsPR1-2) genes were genome-widely identified from Cymbidium orchids. Sequence and phylogenetic analysis revealed that CsNPR1-1 and CsNPR1-2 were grouped closest to NPR1 homologs in Zea mays (sharing 81.98% identity) and Phalaenopsis (64.14%), while CsNPR1-3 was classified into a distinct group with Oryza sativa NPR 3 (57.72%). CsPR1-1 and CsPR1-2 were both grouped closest to Phalaenopsis PR1 and other monocot plants. Expression profiling showed that CsNPR1 and CsPR1 were highly expressed in stem/pseudobulb and/or flower. Salicylic acid (SA) and hydrogen peroxide (H2O2) significantly up-regulated expressions of CsNPR1-2, CsPR1-1 and CsPR1-2, while CsNPR1-3, CsPR1-1 and CsPR1-2 were significantly up-regulated by abscisic acid (ABA) or salinity (NaCl) stress. In vitro transcripts of entire Cymbidium mosaic virus (CymMV) genomic RNA were successfully transfected into Cymbidium protoplasts, and the CymMV infection up-regulated the expression of CsNPR1-2, CsPR1-1 and CsPR1-2. Additionally, these genes were transiently expressed in Cymbidium protoplasts for subcellular localization analysis, and the presence of SA led to the nuclear translocation of the CsNPR1-2 protein, and the transient expression of CsNPR1-2 greatly enhanced the expression of CsPR1-1 and CsPR1-2. Collectively, the CsNPR1-2-mediated signaling pathway is SA-dependent, and confers to the defense against CymMV infection in Cymbidium orchids.

Identification of (Z)-8-Heptadecene and n-Pentadecane as Electrophysiologically Active Compounds in Ophrys insectifera and Its Argogorytes Pollinator.

Sexually deceptive orchids typically depend on specific insect species for pollination, which are lured by sex pheromone mimicry. European Ophrys orchids often exploit specific species of wasps or bees with carboxylic acid derivatives. Here, we identify the specific semiochemicals present in O. insectifera, and in females of one of its pollinator species, Argogorytes fargeii. Headspace volatile samples and solvent extracts were analysed by GC-MS and semiochemicals were structurally elucidated by microderivatisation experiments and synthesis. (Z)-8-Heptadecene and n-pentadecane were confirmed as present in both O. insectifera and A. fargeii female extracts, with both compounds being found to be electrophysiologically active to pollinators. The identified semiochemicals were compared with previously identified Ophrys pollinator attractants, such as (Z)-9 and (Z)-12-C27-C29 alkenes in O. sphegodes and (Z)-9-octadecenal, octadecanal, ethyl linoleate and ethyl oleate in O. speculum, to provide further insights into the biosynthesis of semiochemicals in this genus. We propose that all these currently identified Ophrys semiochemicals can be formed biosynthetically from the same activated carboxylic acid precursors, after a sequence of elongation and decarbonylation reactions in O. sphegodes and O. speculum, while in O. insectifera, possibly by decarbonylation without preceding elongation.

Orchid seed sensitivity to nitrate reflects habitat preferences and soil nitrate content.

Orchids are distributed around the world, however, the factors shaping their specific distribution and habitat preferences are largely unknown. Moreover, many orchids are at risk of becoming threatened as landscapes change, sometimes declining without apparent reason. One important factor affecting plant distribution is nutrient levels in the environment. Nitrates can inhibit not only orchid growth and persistence, but also seed germination. We used in vitro axenic cultures to exactly determine the germination sensitivity of seven orchid species to nitrates and correlated this with soil properties of the natural sites and with the species' habitat preferences. We found high variation in response to nitrate between species. Orchids from oligotrophic habitats were highly sensitive, while orchids from more eutrophic habitats were almost insensitive. Sensitivity to nitrate was also associated with soil parameters that indicated a higher nitrification rate. Our results indicate that nitrate can affect orchid distribution via direct inhibition of seed germination. Nitrate levels in soils are increasing rapidly due to intensification of agricultural processes and concurrent soil pollution, and we propose this increase could cause a decline in some orchid species.

Cryopreservation of Orchids - A Review.

BACKGROUND: The orchids are one of the beautiful creations of nature which stand apart from any other assemblage of flowering plants. They are highly evolutionary and ecologically significant group of plants that have effectively occupied almost every habitat on the earth. Indiscriminate collections and extermination of their natural habitats have threatened many species of orchids with extinction, resulting in a severe reduction of their genetic resources in nature according to recent patents. It is necessary to adopt sound scientific protocols for the preservation of orchid species. METHOD: This cost-effective technique provides large storage time for the conservation of germplasm. Presently, efforts have been made to explore various cryopreservation techniques utilized so far and factors affecting the longevity of the propagules (in vivo and in vitro) while cryopreserving them. The sample to be cryopreserved is freeze-preserved in two ways, a) stepwise at two different subzero temperatures and b) in the rapid method, the samples are placed directly in the liquid nitrogen. RESULTS: The orchid seeds and pollen are the most suitable propagules for cryopreservation of orchids due to their minute size and less space requirement. CONCLUSION: Among the tissues (such as seeds, pollen, protocorms etc.) seeds are the most reliable. The present article reviews the cryopreservation techniques and factors effecting the cryopreservation, for in vitro conservation of orchid gene pool.

Impact of Nitrate and Ammonium ratio on Nutrition and Growth of two Epiphytic Orchids.

Phalaenopsis and Dendrobium do not grow and flower well with 100% ammonium (NH4-N); and there are detailed studies on the effects of nitrate (NO3-N) and ammonium ratios on the flowering, but no information about accumulation of other nutrients and the effects of ammonium toxicity on orchids. For this reason, two experiments were carried out with orchids: Phalaenopsis 'Golden Peoker' and Dendrobium 'Valentine'. Six months after acclimatization the plants were transplanted to individual plastic vessels and the treatments consisted of five ratios (%) of nitrate / ammonium (0/100, 25/75, 50/50, 75/25, 100/0). The sources of NO3-N and NH4-N were calcium nitrate and ammonium sulfate, respectively. After 12 months treatment, when the plants were beginning to issuance of flower stem, the accumulation of: N, P, K, Ca and Mg in the shoot and biometric variables were evaluated for both species. The NH4-N ratio of 40% and 50% of the total nitrogen benefited the growth of Phalaenopsis and Dendrobium, respectively. The application of higher proportions of ammonium resulted in decreased N, K, Ca and Mg absorption, index of green color and increased leakage of electrolytes in Phalaenopsis and Dendrobium. NH4-N proportions greater than 75% for 12 months caused toxicity in Phalaenopsis and Dendrobium.

Development of a PVS2 droplet-vitrification cryopreservation technique for Aranda Broga Blue orchid protocorm-like bodies (PLBs).

　　BACKGROUND: Conservation of commercially important ornamental plants is important to maintain its unique beauty to cater the market demands. OBJECTIVE: The main objective is to develop an efficient cryopreservation technique for Aranda Broga Blue orchid PLBs using droplet-vitrification method. MATERIALS AND METHODS: Several critical factors in cryopreservation were accessed such as preculture concentrations and durations, choice of vitrification solutions, two-step or three-step vitrification, growth recovery medium and PVS2 exposure duration. RESULTS: The best growth regeneration percentage (5%) was obtained when 3-4mm PLBs were precultured in 0.2M sucrose for 3 days, followed by osmoprotection for 20 minutes, dehydration in PVS2 for 20 minutes at 0 degree C, LN storage, thawed and unloading for 20 minutes, and growth regeneration in VW10 medium. PLBs were found to be very sensitive to osmotic stress imposed by high molecular weight cryoprotectant such as sucrose and glycerol. Osmotic potential of growth recovery medium is one of the main factors that affect growth recovery in cryopreserved PLBs. CONCLUSION: Current report showed possibilities in cryopreserving Aranda Broga Blue PLBs using droplet-vitrification technique. However, further improvement of growth recovery can be done by focussing on approaches that facilitate sufficient water removal from PLBs without causing severe osmotic injuries to the plant cells.

Fungal and plant gene expression in the Tulasnella calospora-Serapias vomeracea symbiosis provides clues about nitrogen pathways in orchid mycorrhizas.

Orchids are highly dependent on their mycorrhizal fungal partners for nutrient supply, especially during early developmental stages. In addition to organic carbon, nitrogen (N) is probably a major nutrient transferred to the plant because orchid tissues are highly N-enriched. We know almost nothing about the N form preferentially transferred to the plant or about the key molecular determinants required for N uptake and transfer. We identified, in the genome of the orchid mycorrhizal fungus Tulasnella calospora, two functional ammonium transporters and several amino acid transporters but found no evidence of a nitrate assimilation system, in agreement with the N preference of the free-living mycelium grown on different N sources. Differential expression in symbiosis of a repertoire of fungal and plant genes involved in the transport and metabolism of N compounds suggested that organic N may be the main form transferred to the orchid host and that ammonium is taken up by the intracellular fungus from the apoplatic symbiotic interface. This is the first study addressing the genetic determinants of N uptake and transport in orchid mycorrhizas, and provides a model for nutrient exchanges at the symbiotic interface, which may guide future experiments.

LONG-TERM CONSERVATION OF PROTOCORMS OF Brassavola nodosa (L) LIND. (ORCHIDACEAE): EFFECT OF ABA AND A RANGE OF CRYOCONSERVATION TECHNIQUES.

BACKGROUND: Populations of Brassavola nodosa have been severely affected by habitat destruction and illegal collecting, and as with the majority of orchid species, it is critical to take action to guarantee their continued survival. OBJECTIVE: The present study aimed to establish protocols for the long-term conservation of protocorms of species. MATERIALS AND METHODS: Four different cryogenic techniques were compared: encapsulation-dehydration (ED), encapsulation-vitrification (EV), encapsulation-dehydration-vitrification (EDV) and vitrification. RESULTS: Preculture of protocorms with ABA was a critical factor in obtaining high percentages of regrowth. With vitrification, 100% regrowth was achieved in five treatments, mainly when protocorms were dehydrated with PVS2 for 120 min. 100% regrowth was also obtained with EDV, where the protocorms were precultured with ABA 5 mg/l for 3 days and incubated with PVS2 for 60 min. With the ED, regrowth of 72% was achieved with the preculture of protocorms with ABA 5 mg/l for the three times of incubation used (3, 6 and 9 days). In the case of EV, 92% regrowth, was recorded when protocorms were precultured for 9 days with ABA 3 mg/l and incubated with PVS2 for 90 min. CONCLUSION: Although regrowth of protocorms was obtained with all the techniques used, the vitrification technique is preferred since it requires less labour and is less costly.

UV-B light contributes directly to the synthesis of chiloglottone floral volatiles.

BACKGROUND AND AIMS: Australian sexually deceptive Chiloglottis orchids attract their specific male wasp pollinators by means of 2,5-dialkylcyclohexane-1,3-diones or 'chiloglottones', representing a newly discovered class of volatiles with unique structures. This study investigated the hypothesis that UV-B light at low intensities is directly required for chiloglottone biosynthesis in Chiloglottis trapeziformis. METHODS: Chiloglottone production occurs only in specific tissue (the callus) of the labellum. Cut buds and flowers, and whole plants with buds and flowers, sourced from the field, were kept in a growth chamber and interactions between growth stage of the flowers and duration and intensity of UV-B exposure on chiloglottone production were studied. The effects of the protein synthesis inhibitor cycloheximide were also examined. KEY RESULTS: Chiloglottone was not present in buds, but was detected in buds that were manually opened and then exposed to sunlight, or artificial UV-B light for ≥5 min. Spectrophotometry revealed that the sepals and petals blocked UV-B light from reaching the labellum inside the bud. Rates of chiloglottone production increased with developmental stage, increasing exposure time and increasing UV-B irradiance intensity. Cycloheximide did not inhibit the initial production of chiloglottone within 5 min of UV-B exposure. However, inhibition of chiloglottone production by cycloheximide occurred over 2 h of UV-B exposure, indicating a requirement for de novo protein synthesis to sustain chiloglottone production under UV-B. CONCLUSIONS: The sepals and petals of Chiloglottis orchids strongly block UV-B wavelengths of light, preventing chiloglottone production inside the bud. While initiation of chiloglottone biosynthesis requires only UV-B light, sustained chiloglottone biosynthesis requires both UV-B and de novo protein biosynthesis. The internal amounts of chiloglottone in a flower reflect the interplay between developmental stage, duration and intensity of UV-B exposure, de novo protein synthesis, and feedback loops linked to the starting amount of chiloglottone. It is concluded that UV-B light contributes directly to chiloglottone biosynthesis. These findings suggest an entirely new and unexpected biochemical reaction that might also occur in taxa other than these orchids.

Physical wounding and ethylene stimulated embryogenic stem cell proliferation and plantlet regeneration in protocorm-like bodies of Phalaenopsis orchids.

Phalaenopsis orchids have been regenerated by inducing protocorm-like bodies (PLBs) from etiolated leaf sections. However, the physiological and molecular mechanisms of secondary PLB development and subsequent proliferation have not been explored. Bisectionally cutting primary PLBs resulted in more secondary PLBs at 5 weeks, suggesting an embryogenic stem cell property imposed by wounding of primary PLB tissues. The ethylene precursors ethephon and 1-aminocyclopropanecarboxylic acid and the ethylene perception inhibitor silver nitrate increased PLB formation, while aminoethoxyvinylglycine decreased PLB formation. Ethylene content in wounded PLB explants increased over culture time in media containing ethylene precursors or inhibitors. mRNA levels of PhACS2, PhACS3, and PhACO were increased by ethephon and decreased by ethylene inhibitors. Expression of genes in the ethylene signaling pathway was enhanced following ethylene-precursor treatment and was mitigated by ethylene inhibitors during PLB proliferation. Transcription of PhETR and PhEIN3, as well as PhERS, PhCTR, and PhGTP, was significantly increased 12 h after ethylene treatment. Ethylene and physical wounding stimulated secondary PLB formation in Phalaenopsis, probably through ethylene biosynthesis and signal transduction.

Mass propagation of Cymbidium giganteum Wall. ex Lindl. using in vitro seedlings.

In vitro seedlings were used as explants for protocorm like bodies (PLBs) production which in turn were used for regeneration purpose. PLBs were induced from the base of seedlings (1.0-1.5 cm in size) in MS + BAP (8.88 microM). After 90 days of inoculation, PLBs production rate started declining and most of the PLBs turned into plantlets. Preculture of seedlings in 1.0 microM thidiazuron (TDZ) for 7 days and transfer to BAP supplemented medium resulted in production of 16 PLBs per seedling within 90 days of culture. Increase of TDZ concentration to 2.5 microM and preculture time 15 days, resulted in induction of highest number of PLBs (19 PLBs per seedling) in the basal medium. The results emphasized the importance of thidiazuron (TDZ) concentration and preculture time for PLBs proliferation from the base of seedlings. The PLBs thus produced were used for regeneration studies. Irrespective of single, segmented or clumps of PLBs, the regeneration response was 100% in 2,4-D (4.52 microM) and KN (4.64 microM) but when KN was replaced by BAP (8.88 microM), response was observed only in clumps of PLBs, whereas in single and segmented ones it was 99 and 97%, respectively. Regenerants developed stout root system in half strength M medium supplemented with 2.84 microM of IAA and transferred to greenhouse with 90% survival. The present study holds tremendous potential as the mother plant is not destroyed and PLBs are produced as a continuous system.

[Residue decline dynamics and safety utilization of carbendazim in cultivation of Anoectochilus roxburghii].

The paper aimed to study the residue decline dynamic and standards for safety utilization of carbendazim in roots, stems, leaves of Anoectochilus roxburghii and in growth media. Samples extracted with methanol were purified by liquid-liquid extraction and analysed by HPLC. The results showed that average rate of recovery was 82.9% - 95.7% and RSD were 2.0% - 6.3% with add of carbendazim in respectively diverse concentration, which meets inspection requirement of pesticide residue. Two kinds of dosages of carbendazim were treated, varying from recommended dosage (1.0 kg x hm(-2)) to 1.5 times recommended dosage (1.5 kg x hm(-2)). Results of two years test showed that the half-life period of carbendazim were 7.01 - 8.51 d in the growth media of A. roxburghii, 3.58 - 4.27 d in stems and 3.50 - 3.91 d in leaves, 4.93 - 5.71 d in roots. Providing max recommended residue of carbendazim in the cultivation of A. roxburghii is 0.5 mg x kg(-1), sprayed 4 times a year with the dosage of 1.0 kg x hm(-2), 28 days is proposed for the safety interval of the last pesticide application's and harvest's date.

A novel in vitro protocol for inducing direct somatic embryogenesis in Phalaenopsis aphrodite without taking explants.

An alternative in vitro protocol for embryo induction directly from intact living seedlings of Phalaenopsis aphrodite subspecies formosana was established in this study. Without the supplementation of plant growth regulators (PGRs), no embryos were obtained from all the seedlings when cultured on the solid medium. In contrast, embryos formed from the seedlings on the 2-layer medium and the 2-step culture system without the use of PGRs. It was found that the age of the seedlings affected embryo induction. The 2-month-old seedlings typically had higher embryogenic responses when compared with the 4-month-old seedlings in the 2-layer medium or 2-step system. For the 2-month-old seedlings, 1 mg/L TDZ resulted in the highest number of embryos at the distal site of the shoot. However, on the leaves' surface, 0.5 mg/L TDZ induced the highest number of embryos. When the 2-month-old seedlings were cultured using the 2-step method at 1 mg/L of TDZ, the highest embryogenic response was obtained, with an average of 44 embryos formed on each seedling. These adventitious embryos were able to convert into plantlets in a PGR-free 1/2 MS medium, and the plantlets had normal morphology and growth.

[Effects of different transplanting conditions on survival rate and growing status of Anoictochilus roxburghii plantlets].

The growing status of Anoectochilus roxburghii seedling was observed and the survival rate of seedlings, height, stem diameter and plant fresh weight under the conditions of different transplanting substrate compositions, planting density, shading rate were measured. The results showed that the effects of different transplanting substrates, planting densities, shading rates and nutrient solutions on the growing status of A. roxburghii plantlets varied greatly. A. roxburghii plantlets demonstrated a high survival rate and better growing status under the Following conditions: the ratio of peat and river sand as 2: 1, the planting density as 3 cm x 3 cm, the shading rate as 70%, and the nutrient solution as 1/4MS. The findings of the study provide a solid technical solution for the artificial cultivation of A. roxburghii plantlets.

Cryopreservation of protocorm-like body-derived shoot tips of Calanthe davidii by droplet-vitrification.

BACKGROUND: Evaluation of the conservation status of Calanthe plants in China indicated that 18 species were endangered or critically endangered. Comprehensive conservation solutions including ex situ methods, are urgently required to protect Calanthe species in China. OBJECTIVE: The study aims to develop a simple and efficient cryopreservation protocol using droplet-vitrification for Calanthe davidii. METHODS: Protocorm-like bodies (PLBs) were induced from nodal sections of in vitro shoots, and their proliferation was promoted by a thin cell layer culture procedure. Shoot tips excised from three leaf-stage PLBs were used in cryopreservation experiments. Key factors of the droplet-vitrification procedure including sucrose preculture, treatment with PVS2 solution and post-rewarming culture conditions were optimized to achieve a high level of regeneration. RESULTS: When the optimized procedure was applied, 77.8 ± 3.9% of cryopreserved shoot tips withstood liquid nitrogen exposure and regenerated into new PLBs. CONCLUSION: These results highlighted the importance of post-rewarming osmo-conditioning for regeneration of cryopreserved shoot tips.

Cryopreservation of Brassidium Shooting Star orchid using the PVS3 method supported with preliminary histological analysis.

Cryopreservation is an alternative, safe, and cost-effective method for long-term plant genetic resource conservation. This study was conducted to optimize the conditions for cryopreserving the protocorm-like bodies (PLBs) of Brassidium Shooting Star orchid with the PVS3 vitrification method. Five parameters were assessed in this study: PLB size, sucrose concentration, preculture duration, PVS3 duration, and unloading duration. The viability of the cryopreserved PLBs was determined using the triphenytetrazolium chloride assay and growth recovery assessments. The optimum condition for the cryopreservation of the PLBs of Brassidium Shooting Star orchid is based on the size range between 3 and 4 mm precultured with half-strength semi-solid MS media supplemented with 0.25 M sucrose for 24 h, followed by treatment with loading solution mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min. The PLBs were then dehydrated with PVS3 at 0 °C for 20 min prior to immersion in liquid nitrogen; finally, the PLBs were immersed with half-strength liquid MS media supplemented with 1.2 M sucrose for 30 min. Histological analyses displayed denser cytoplasm and voluminous nucleus in the cryopreserved PLBs of Brassidium Shooting Star orchid.

Composition of Cypripedium calceolus (Orchidaceae) seeds analyzed by attenuated total reflectance IR spectroscopy: in search of understanding longevity in the ground.

PREMISE OF THE STUDY: Orchid seeds are minute and covered with a thin coat, yet they often have a long life after dispersal. They are notorious for low and irregular germination, in nature as well as in vitro. Since orchids are often rare species of conservational and commercial interest, reproduction by seeds is an important concern. The purpose of this study was to learn more about the resilience of these highly specialized seeds and stimulatory processes toward germination. • METHODS: We studied testa and embryos of Cypripedium calceolus to identify natural components in intact seeds and the impact of 7 yr in soil in its natural habitat. We also analyzed the effects of Ca(OCl)2, used technically to enhance germination for cultivation in vitro. For the first time with this kind of plant material, we used attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, an ideal method for minute sample sizes and surface selectivity. Thus, we could link treatments with changes in seed surface chemistry. • KEY RESULTS: A lignin-like polymer is an essential testa component that undergoes degradation by soil or hypochlorite processes. In both cases, we found a build-up of CaCO3 on the testa, which could interact with lignin to enhance germination. Very minor changes occurred in embryo reserve nutrient content after a long sojourn underground, which supports their continued viability. • CONCLUSIONS: We suggest that degradation of lignin and enrichment of the testa surface with CaCO3 are important stimulants of germination both in the habitat and during laboratory sowing.

Cryopreservation of Cyrtopodium hatschbachii Pabst (Orchidaceae) immature seeds by encapsulation-dehydration.

The aim of the present study was to investigate the efficiency of the encapsulation-dehydration technique for cryopreservation of Cyrtopodium hastchbachii Pabst seeds. Immature seeds of this species were cryopreserved by an encapsulation-dehydration technique. Seeds of five immature pods, 120 days after pollination, were encapsulated in 3% calcium alginate matrix and pretreated in liquid medium supplemented with 0.08 M sucrose (24 h), 0.15 M sucrose (24 h), 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h) in shaker at 60 rpm. Alginate beads were dehydrated 5 h in silicagel and immersed in liquid nitrogen for 12 h. Cryopreserved beads were thawed at 30 degrees C for 1 min, rehydrated using the same liquid mediums [0.75 M sucrose (24 h), 0.5 M sucrose (24 h), 0.25 M sucrose (48 h) and 0.15 M sucrose (24 h)] and cultivated in half strength Murashige & Skoog medium (1962) with the addition of 2 g/L activated charcoal. Sixty four percent of seeds survived and developed into acclimatized plants after being cryopreserved. In this work, the encapsulation-dehydration technique was employed for first time in Cyrtopodium hatschbachii.