Asymbiotic Germination Research Articles

Comparative Growth Media Performances on in vitro Propagation of Some Salep Orchids

Due to the increasing demand and over collection of orchids from nature to produce salep, scientists have been led to search for more efficient ways to propagate these specific orchids in vitro . This present study compares germination performances of two commercial (Orchimax and Knudson C) and one specially prepared orchid growth media (SV), on economically and medicinally important orchids used to make salep; ( Anacamptis pyramidalis , Dactylorhiza romana , Neotinea tridentata and Anacamptis morio subsp. morio ) and further aims to obtain a mature orchid plant by following the natural environmental cycle. Significant differences in seed germination and protocorm development were observed. Asymbiotic germination tests showed that the specially prepared growth media performed better than the commercial media by 79.11% germination rate. Also, that A. morio subsp. morio had the best germination rate by 88.91%. Protocorms developed in the sixteenth week after sowing. Soil was collected from the natural habitat of each species and was used as a potting substrate, and this helped orchids to pass their initial acclimatization stage. Regeneration success of orchids at in vitro conditions could be increased by using SV growth medium, following their natural hardening off cycle and using specific substrates from their respective habitats.

ESTIMULACIÓN DE LA GERMINACIÓN Y DESARROLLO in vitro DE Laelia autumnalis CON RAYOS GAMMA

La germinación natural de las semillas de la orquídea L. autumnalis es baja, porque requiere condiciones específicas del árbol hospedero y factores ambientales favorables. La germinación asimbiótica in vitro es un método de propagación eficiente; sin embargo, el desarrollo de las plántulas requiere de uno a dos años. El objetivo fue evaluar el efecto de la radiación gamma 60Co para estimular la germinación de semillas y el desarrollo in vitro de plántulas de L. autumnalis. Se irradiaron 22 frutos a diferentes dosis (3 a 30 Gy, con intervalos de 3 Gy), además de un tratamiento sin irradiar utilizado como testigo. Las semillas se cultivaron en medio Murashige y Skoog sin fitohormonas. El diseño experimental fue completamente al azar con 16 a 32 repeticiones; la unidad experimental fue un frasco con 20 mg de semillas. A los cinco días y posteriormente cada 10 días se cuantificó el número de semillas en las etapas de imbibición, formación de protocormos fotosintéticos, protocormos en diferenciación, desarrollo de promeristemos, hojas y plántulas. Se realizó un análisis de varianza y prueba de Tukey (α = 0.05). Las semillas irradiadas entre 3 y 15 Gy formaron 61 % más protocormos fotosintéticos que las semillas sin tratar. Las semillas irradiadas con 3, 15 y 18 Gy formaron 73 % más promeristemos. Todos los protocormos tratados con 3 Gy formaron hojas 60 días después de la siembra (dds); en contraste, sólo 12.5 % de las semillas no tratadas formaron hojas. El 66.5 % de las semillas tratadas con 15 Gy desarrollaron plántulas a los 80 dds. Se confirmó el efecto radioestimulante de la radiación gamma a dosis bajas; con 3 Gy se formaron promeristemos, hojas y plántulas completas 20, 20 y 10 días, respectivamente, antes que en el tratamiento control. La radiación a dosis superiores fue negativa al retrasar la germinación y el desarrollo de plántulas.

In vitro asymbiotic germination for micropropagation of the recalcitrant terrestrial orchid Chloraea crispa (Orchidaceae).

Premise of the study:Chloraea crispa is a terrestrial Orchidaceae species native to Chile, characterized by a beautiful and showy inflorescence. The species has a great potential for commercial exploitation in the cut flower industry, but it is essential to improve propagation methods to avoid endangering its natural populations. Because this species is hard to propagate using traditional greenhouse techniques, in vitro techniques offer an effective tool for its large-scale production in terms of germination, growth, and propagation.Methods:The current study evaluated the effect of the culture medium on the asymbiotic germination of C. crispa seeds, as well as the effects of the plant growth regulators 6-benzylaminopurine and indole-3-butyric acid. Different light regimes were also studied.Results:A significant effect was observed for the interaction between culture media and light regime on the morphogenic response of the seeds. The highest rate of embryonic germination was obtained in Van Waes medium supplemented with 0.1 mg·L−1 of 6-benzylaminopurine.Discussion:For the first time, asymbiotic culture of this species using biotechnology tools has been developed. Plantlets developed very well under in vitro conditions, allowing the possibility to propagate and store genetic material for conservation and domestication purposes.

ITRAQ and RNA-Seq Analyses Provide New Insights into Regulation Mechanism of Symbiotic Germination of Dendrobium officinale Seeds (Orchidaceae).

Mycorrhizal fungi colonize orchid seeds and induce germination. This so-called symbiotic germination is a critical developmental process in the lifecycle of all orchid species. However, the molecular changes that occur during orchid seed symbiotic germination remain largely unknown. To better understand the molecular mechanism of orchid seed germination, we performed a comparative transcriptomic and proteomic analysis of the Chinese traditional medicinal orchid Dendrobium officinale to explore the change in protein expression at the different developmental stages during asymbiotic and symbiotic germination and identify the key proteins that regulate the symbiotic germination of orchid seeds. Among 2256 identified plant proteins, 308 were differentially expressed across three developmental stages during asymbiotic and symbiotic germination, and 229 were differentially expressed during symbiotic germination compared to asymbiotic development. Of these, 32 proteins were coup-regulated at both the proteomic and transcriptomic levels during symbiotic germination compared to asymbiotic germination. Our results suggest that symbiotic germination of D. officinale seeds shares a common signaling pathway with asymbiotic germination during the early germination stage. However, compared to asymbiotic germination, fungal colonization of orchid seeds appears to induce higher and earlier expression of some key proteins involved in lipid and carbohydrate metabolism and thus improves the efficiency of utilization of stored substances present in the embryo. This study provides new insight into the molecular basis of orchid seed germination.

Asymbiotic germination, multiplication and development of Alatiglossum fuscopetalum (Orchidaceae) as affected by culture medium, sucrose and growth regulators

Germinacao assimbiotica multiplicacao e desenvolvimento de Alatiglossum fuscopetalum (Orchidaceae) influenciados pelo meio de cultura, sacarose e reguladores de crescimento. Este trabalho teve como objetivo estudar a germinacao e o desenvolvimento in vitro de Alatiglossum fuscopetalum (Hoehne) Baptista. Foram avaliados os efeitos dos meios Murashige/Skoog (MS e ½MS), Knudson (KN), e Vacin/ Went (VW) na germinacao. Analisou-se tambem a infl uencia desses meios na multiplicacao e crescimento de plântulas com 90 dias de idade, e de diferentes concentracoes de sacarose, benziladenina [BA] e acido indolilbutirico [AIB] na multiplicacao e crescimento de plantas com 120 dias de idade. O meio ½MS proporcionou a maior germinabilidade. O meio MS foi superior aos demais em relacao ao crescimento de plântulas de 90 dias, enquanto que o ½MS foi o mais efi caz para a multiplicacao e crescimento de plantas de 120 dias. Concentracoes de 1% a 3% de sacarose favoreceram o crescimento. Para a proliferacao de brotos e crescimento radicial os melhores tratamentos foram 1 mg L-1 de BA e 2 mg L-1 de AIB, respectivamente.

In vitro culture of Chloraea gavilu Lindl., an endemic terrestrial orchid from Chile

Orchids are the second most diverse plant family, recognized for their importance as ornamental species; this has driven research development in propagation. One of the most common culture methodologies is in vitro asymbiotic germination, in which the nutritional conditions that provide orchids with a fungal partner are emulated. Although Chile possesses more than 60 terrestrial orchid species, in vitro cultivation protocols have only been developed for Chloraea crispa. In Southern Chile, Chloraea gavilu stands out due to its floral characteristics. We evaluate different explants and cultivation conditions for C. gavilu. The best germination and development results were achieved in the MM medium +0.1% yeast extract +1% sucrose +0.454 µmol l−1 TDZ, using immature seeds of 24–30 days after pollination, which we cultivated into seedlings in order to be acclimatized and mycorrhized. In addition, induction of protocorm-like bodies was achieved from germinated seeds, using the same culture media as in the germination and development of immature C. gavilu seeds. This resulted in the successful asymbiotic germination of immature seeds, along with the micropropagation of a terrestrial, temperate orchid. We hope to use our protocol in the commercial production of Chilean orchid species as well as to propagate threatened species.

Asymbiotic germination in three Chloraea species (Orchidaceae) from Chile

ABSTRACT Orchids require symbiotic fungi and/or specific conditions to germinate. Asymbiotic techniques have been shown successful for orchid germination. In Chile, Chloraea include many endemic, and potentially ornamental, terrestrial orchid species. In this study, individuals of Chloraea crispa, C. gavilu and C. virescens were manually autopollinated. The resulting capsules were sterilized and seeds were aseptically obtained. We evaluated asymbiotic germination in: Agar Water (AW), Knudson C (KC), Banana Culture Media (CMB), Tomato Culture Media (CMT), Malmgren Modified (MM), Murashige and Skoog (MS), and MS modified (MS1/2). Seeds were incubated in the dark at 24±1 oC for 2 weeks. Then they were put in 16/8 h light/dark cycles for 14 weeks. We registered germination and embryo development in the different culture media. After 8 weeks, all tested Chloraea species germinated in most culture media. After 16 weeks, embryos in MM showed an evident shoot. In AW only the pre-germination stage was achieved. In C. gavilu germination was over 90% in MM, CMT, and CMB. In KC, a lower germination was obtained in all tested species compared to the other culture media, except for AW. Asymbiotic germination of endemic Chilean orchids could be a viable strategy for their conservation and propagation as ornamental species.

Seed storage and asymbiotic germination of Hadrolaelia grandis (Orchidaceae)

Hadrolaelia grandis is an endangered species of orchid that is experiencing population declines due to habitat destruction and collection for ornamental purposes. Seed storage, in vitro germination and development of seedlings for propagation were investigated. Tetrazolium test (TZ) and asymbiotic germination were used to evaluate seeds stored at −20°C and −80°C for 24, 30, and 36months. Seeds were germinated on Woody Plant Medium (WPM), Murashige & Skoog (MS), Vacin & Went (VW) and Knudson C (KC) media. The temperature of −80°C was the most effective for seed storage since the percentage of viable seeds (81.86%), as determined by TZ, was higher than those stored at −20°C (64.24%) for 36months. WPM proved to be the most effective medium for seed germination and seedling development (94.71%, and a germination speed index of 25.33) after 36months of storage at −80°C. Protocorm elongation and root development were achieved on WPM medium containing activated charcoal (1gL−1), and after 16weeks seedlings were successfully acclimatized after 12weeks (91.07% survival rate) using vermiculite as a substrate.

In vitro reproduction of three Limodorum species (Orchidaceae): impacts of scarification methods and nitrogen sources on mature seed germination and seedling development

Limodorum species are hard-to-cultivate, germinating with difficulty or not germinating at all on a number of media. The present work was focused on the effects of seed sterilization-scarification methods and of different nitrogen sources on in vitro seed germination and seedling development of Limodorum abortivum, L. brulloi and L. trabutianum. Our results showed that sodium hypochlorite solution with a 1% concentration of available chlorine was ineffective for both sterilization and scarification of Limodorum seeds, while a 5% concentration was found to be an effective sterilizing and scarifying solution for L. trabutianum. The positive effect of organic nitrogen in promoting asymbiotic germination in many terrestrial orchids was confirmed by this study for L. trabutianum and L. brulloi, which showed significantly higher germination percentages on Basal Medium modified than on Knudson C, which contains inorganic nitrogen. This is the first report of in vitro asymbiotic germination of mature seeds of L. brulloi and L. trabutianum, especially successful for L. trabutianum that showed high germination percentages (>63%) and an active seedling development. These results may be useful for conservation purposes, since L. brulloi is a narrow Italian endemics and L. trabutianum is threatened with extinction both in Italy and in France.

Seasonal dynamics of mycorrhizal fungi in Paphiopedilum spicerianum (Rchb. f) Pfitzer — A critically endangered orchid from China

Paphiopedilum spicerianum (Rchb. f) Pfitzer has only one wild population with 38 individuals in Pu’er, southern China, and has been closely monitored under the Plant Species with Extremely Small Population project launched in 2005. Immediate conservation actions, including ex situ conservation, pollination observations, and studies of asymbiotic germination, reintroduction and relocation to new habitats have been conducted because the single existing population is threatened by habitat degradation. In this study, next generation sequencing by Illumina MiSeq with primer pair ITS-3 (F) and ITS-4OF (R) amplified ITS 2 region was conducted to identify and explore the associated uncultivatable fungal community structure and dynamics through seasonal and environmental changes. Assessments of the entire fungal communities from different seasons and habitats indicated that the communities were sensitive to changes in habitat but not to seasonal changes. Between plant roots and soil, two independent fungal communities and compartmentalization of microhabitats were detected. Hydrolysable nitrogen, total phosphorous, and water content were the most significant factors for fungal communities. Fungal community change could be related to nutrient requirements and plant phenology. Paphiopedilum spicerianum can associate with a wide range of orchid mycorrhizal fungal genera and is capable of utilizing different genera simultaneously. The low specificity to mycorrhizal fungi in adult Paphiopedilum spicerianum suggests that the choice of locations for new populations might be broader than initially anticipated.

Non-specific symbiotic germination of Cynorkis purpurea (Thouars) Kraezl., a habitat-specific terrestrial orchid from the Central Highlands of Madagascar.

Orchids, particularly terrestrial taxa, rely mostly on basidiomycete fungi in the Cantharellales and Sebacinales that trigger the process of seed germination and/or initiate the full development of the seedling. During the course of development, orchids may associate with the same fungus, or they may enlist other types of fungi for their developmental needs leading to resilience in a natural setting. This study examined in vitro seed germination and seedling developmental behavior of Cynorkis purpurea, a terrestrial orchid from the Central Highlands of Madagascar. This species is mostly restricted to gallery forests in the Itremo Massif, in moist substrate between rocks bordering streams. The main objective was to understand the influence of diverse mycorrhizal fungi on seed germination and further development of C. purpurea. The study aims to compare symbiotic versus asymbiotic germination and seedling development with seeds and fungi collected from a 13-km(2) area in the Itremo region. Seeds collected from the wild were sown with diverse orchid mycorrhizal fungi (OMF) spanning 12 operational taxonomic units (OTUs) in three genera (Tulasnella, Ceratobasidium, and Sebacina) acquired from different habitats. Treatments were assessed in terms of the percentage of germinated seeds and fully developed seedlings against those in asymbiotic control media treatments. Overall, OMF significantly improved seedling development within the 12-week experiment period. Sebacina as a genus was the most effective at promoting seedling development of C. purpurea, as well as having the ability to enter into successful symbiotic relationships with orchids of different life forms; this new knowledge may be especially useful for orchid conservation practiced in tropical areas like Madagascar. A Sebacina isolate from an epiphytic seedling of Polystachya concreta was the most effective at inducing rapid seedling development and was among the five that outperformed fungi isolated from roots of C. purpurea. C. purpurea was found to be a mycorrhizal generalist, despite its specific habitat preference, highlighting the complex interaction between the plant, fungi, and the environment. The potential impact on conservation strategies of understanding the requirements for orchid seed germination and development by identifying and using OMF from diverse sources is discussed in detail.

Highly compatible Epa-01 strain promotes seed germination and protocorm development of Papilionanthe teres (Orchidaceae)

Symbiotic seed germination serves as a preferred method for orchid multiplication related to conservation and reintroduction programs, which involves isolation, identification, and germination-enhancing testing of symbiotic fungi. This study uses seeds of Papilionanthe teres, a locally endangered and medicinally valuable epiphytic orchid, to attract germination-enhancing fungi on its four host plants. Only one common and highly compatible fungus (Epa-01 strain, Epulorhiza sp.), isolated from seed baiting near three host plants (Averrhoa carambola, Lagerstroemia villosa, Callistemon rigidus), enhanced seed germination by more than 80 % and promoted protocorm development to reach stage 5 (with two leaves). Seeds cocultured with the Epa-01 strain and oat meal agar medium significantly outperformed in germination and growth speed compared with those cocultured with asymbiotic germination medium only, indicating that symbiotic seed germination is an effective method for P. teres seedling production. Bark substrate types have profound effects on symbiotic seed germination and protocorm development possibly due to different abundance and growth vitality of the Epa-01 strain on the four host plants. A significant difference was found in the developmental speed of symbiotic seeds between A. carambola and the other three host plants under ex situ and ex vitro seed germination treatments (all P < 0.05). The results suggest that in situ seed baiting may be used to effectively capture germination-enhancing fungi in epiphytic orchids, and testing the effects of bark substrate types on seed germination and protocorm development contributes to selecting appropriate host plants for its reintroduction into natural habitats.

Protocols for In Vitro Mass Multiplication and Analysis of Medicinally Important Phenolics of a Salep Orchid, Satyrium nepalense D.Don ("Salam Mishri").

Satyrium nepalense is a rare and threatened medicinal orchid, populations of which in its native habitats are dwindling because of indiscriminate collections and habitat destruction, thus necessitating the development of methods for its in situ and ex situ conservation. Because of non-endospermous nature of the seeds and the immature embryos at seed dispersal stage, orchids cannot be seed-propagated as other plants. Micropropagation, using plant tissue culture techniques, offers an effective method for the multiplication of orchids. In this chapter, a five-step efficient reproducible protocol for large-scale in vitro multiplication of Satyrium nepalense is described. The first step involves asymbiotic germination of seeds isolated from immature green pods and cultured on Mitra's medium (M) gelled with 0.8 % agar and supplemented with 2 % sucrose and 1 % peptone (hereafter referred to as basal medium, BM). On this medium, seeds start germinating after a week of culture. Protocorms developed from the seeds are sub-cultured on BM fortified with 4 μM kinetin (Kn) after 8 weeks, for shoot differentiation and multiplication. The shoots developed on Kn-supplemented medium are transferred to BM alone for their elongation for the same period. The elongated shoots are transferred to the rooting medium, comprising BM supplemented with 0.5 or 1.0 μM indole-3-butyric acid, for further 8 weeks. The regenerated plantlets are transferred to a potting mix of sand and vermiculite (1:1) for acclimatization. The tubers and leaves excised from both in vitro-developed plants and those from their native habitats are analyzed and compared for the contents and concentration of medicinally important phenolics using high-performance liquid chromatography (HPLC), details of which are provided in this chapter.

A cryopreservation protocol for ex situ conservation of terrestrial orchids using asymbiotic primary and secondary (adventitious) protocorms

In a bid to better conserve endangered terrestrial orchids, we detail cryogenic research using a widely distributed terrestrial orchid, Caladenia latifolia, as a model species for development of cryopreservation for primary (seed generated) and secondary (adventitious) protocorms. Primary protocorm cryopreservation (using droplet vitrification) involved a number of experimental lines of inquiry: investigation of a suitable plant vitrification solution (PVS) by comparing three variants of a standard PVS (2, 3 and 4), determining the most suitable primary protocorm developmental stage for successful cryopreservation, testing the effectiveness of a preculture medium treatment prior to cryopreservation, and investigating temperature preconditioning at the preculture stage as well as different components of the recovery medium. Primary protocorms were generated using asymbiotic in vitro germination media developed by the authors specifically for the test species (half-strength MS macroelements and microelements with 5% (v/v) fresh filter sterilized coconut water). Secondary protocorms were propagated using an in vitro proliferation medium (½ MS with 5 μM α-naphthaleneacetic acid + 2 μM 6-benzylaminopurine). A modified preconditioning step was developed, involving incubation on ½ MS with 0.2 M raffinose for 48 h at 15°C instead of 20°C. The standard recovery medium (½ MS 1 μM zeatin + 0.5 μM gibberellic acid) was replaced after the first week following warming from liquid nitrogen (LN), with asymbiotic germination medium (½ MS + 5% (v/v) coconut water) for the remainder of the recovery phase. This new step increased the survival of primary protocorms from 68 to 85%. The average post-cryostorage regeneration of plants from primary protocorms increased from 17 to 48% after a 6-wk incubation. A similar protocol increased the survival of secondary protocorms from 63 to 84%. Regeneration of plants from secondary cryostored protocorms increased from 11 to 26% after 14 wk. The protocols developed here provide a useful template for advancing cryoconservation of other orchid taxa, particularly rare and threatened species.

In vitro seed germination and seedling growth of Calanthe discolor Lindl

Abstract We investigated the effects of sodium hypochlorite (NaOCl) and culture medium on embryo swelling and germination of Calanthe discolor Lindl., and established a method for determining the swelling and protocorm formation of C. discolor seeds via in vitro examination of immature seeds. Treatment of immature seeds with NaOCl greatly enhanced the extent of embryo swelling and protocorm formation of immature zygote embryos compared to seeds without NaOCl treatment. The effects of the culture media were also evaluated with regard to embryo swelling and protocorm formation of in vitro cultured seeds with and without NaOCl treatment. Additionally, the effects of white fluorescent light and red and blue LED lights on seedling growth in in vitro culture were examined. The most suitable condition for seedling growth after 12 weeks of culture was the red LED light with POM medium. These results show effective asymbiotic germination and growth of C. discolor.

Asymbiotic germination and seedling growth of Calanthe striata f. sieboldii Decne. ex Regel

Calanthe striata f. sieboldii Decne. ex Regel is a terrestrial orchid with beautiful flowers arranged in racemose inflorescences. This species is threatened due to over-collection and loss of suitable habitats. Asymbiotic germination is useful in the conservation efforts to re-establish plants in the wild, and for commercial propagation. In this study, we investigate the effects of sodium hypochlorite (NaOCl), three types of culture media (Phytomax Orchid Maintenance - POM, Seed Germination Maintenance - SGM, and Murashige and Skoog 1962 - MS), and plant growth regulators on embryo swelling, protocorm formation, and embryo diameter of C. striata f. sieboldii. Treatment with 1% NaOCl for 30 min greatly enhanced embryo swelling (28.3%), embryo diameter (), and embryo protocorm formation (54.8%) compared to seeds without NaOCl treatment (embryo swelling 8.5%, embryo diameter , and protocorm formation 13.4%) on POM medium. Protocorm formation on POM medium supplemented with 1.0 mg/L N6-benzyladenine (BA) (95.6%) was better than the control (54.5%). Additionally, the effects of activated charcoal (AC) and sucrose on seedling growth in in vitro culture were examined. The protocorm converted into healthy plants with well-developed shoot primordia on the POM medium with AC and sucrose. The most suitable conditions for seedling growth after 10 weeks of culture were the POM medium with AC or sucrose. These results show effective asymbiotic seed germination and in vitro seedling growth of C. striata f. sieboldii.

Desarrollo de cápsulas y germinación in vitro de Phragmipedium humboldtii, P. longifolium y P. pearcei

Slipper orchids belonging to the genus Phragmipedium (subfam. Cypripedioideae) are seriously threatened and therefore listed in Appendix I of CITES. Less research has been conducted in this genus than in others belonging to the same subfamily. In this work, we evaluated development of capsules (seed pods) from Phragmipedium humboldtii, P. longifolium and P. peaarcei from the time of pollination until opening. Moreover, seed viability was tested with the tetrazolium method in each of the capsules that were subsequently used to evaluate the effect of light and two culture media (Knudson C vs. Murashige and Skoog half concentrated) on in vitro asymbiotic germination and seedling growth. 100% of the pollinated flowers developed capsules, which differentiated in terms of length and diameter among species. While the length of the capsules remained constant during development, their diameter increased during the first 6-8 weeks and then stopped. Time required for maturity and opening of the capsules also varied among species (31 weeks in P. humboldtii, 16 weeks in P. longifolium and 9.5 weeks in P. percei). Seed viability differed among species as well, averaging 34.3% in P. humboldtii, 44.7% in P. longifolium and 82.3% in P. pearcei. Furthermore, seed viability of each capsule was used to adjust the germination rate measured in each case. While very few P. humboldtii viable seeds germinated under the conditions tested (2.9%), better results were observed in the other two species (close to 40% germination). No significant effect of light/darkness regime or of culture medium was observed on germination. However, better growth of the germinated embryos was observed with the Knudson C medium and darkness conditions. Further subculture of the gro- wing plantlets under light conditions induced development of roots and allowed successful acclimatization of seedlings in the greenhouse.

Asymbiotic Germination of Immature Seeds in an Ornamentally Important'Fox-tail ’Orchid,Aerides multifloraRoxb.

Present paper deals with asymbiotic ger-mination potential of immature seeds in Aeridesmultiflora Roxb. for its rapid mass propagation in vitro. Seeds were inoculated on Potato Dex-trose Agar (PDA) medium with and without growth adjuncts (CH, Casein Hydrolysate; P, Pep-tone, YE, Yeast Extract; each 0.1%) and activated charcoal (AC; 0.2%). Seed germination initiated after 1.20±0.42 weeks (wks) on CH supplement-ed medium. The percent germination response showed significant differences in majority of combinations used; it was lowest (48.00±2.16) in basal medium and highest (99.50±0.85) in medi-um supplemented with CH and AC. Protocorm formation started after 4.15±0.48 wks of cultur-ing on CH and AC containing medium. First leaf and first root differentiated after 7.30±0.82 and 14.00±0.67 wks of culturing respectively on me-dium supplemented with P and AC. Seedlings with 3–4 leaves and 2–3 roots were obtained af-ter 18.30±0.67 wks. The medium supplemented with CH and AC proved to be the best for seed germination, protocorm formation and multipli-cation, and that containing P and AC for early leaf and root differentiation, and seedling devel-opment. The seedlings were subjected to hard-ening and 72.56±3.18% of them survived under greenhouse conditions.

In vitropropagation 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.

Asymbiotic Germination and Seedling Development of Dimorphics lowii (Orchidaceae)

Dimorphorchis lowii, a threatened Borneo endemic epiphytic orchid, is gradually becoming rare due to over collecting and habitat disturbance. Therefore this study was carried out to obtain in vitro propagation through asymbiotic seed germination and seedling development by optimizing capsule maturity, nutrients requirements and light conditions before introduce back to its natural habitat for conservation purposes. Capsules were collected at 100, 150 and 170 days through hand-pollination. The seeds were germinated on Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW) media added with 10% additives (coconut water, potato homogenate and tomato juice) under light and dark conditions. Seeds from 150 days old capsule grow on VW medium added with potato homogenate under light condition were observed to be an optimum condition with higher germination percentage as compare to other treatments. Seeds started to germinate by swollen of embryo (8 days) before the testa were ruptured at 23 days and further developed into mature protocorm at 33 days. Seeds with 4 leaves and 5 roots were ready for hardening process within 200 days. A successful developed system for in vitro propagation will contribute to the development of a sustainable management program for D. lowii in Sabah, Malaysia.