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Orchid seed germination and seedling culture - A manual
... Knudson (1922) demostró que en ausencia de hongos era posible la germinación de semillas de orquídeas en un medio simple, rico en minerales y azúcares. Este investigador además encontró que las semillas de algunos géneros, entre las que se encuentran Cattleya y Epidendrum, germinaban en forma asimbiótica en condiciones in vitro; no obstante se estimó que no siempre es posible desarrollar un medio de cultivo en el cual una determinada orquídea pueda germinar y desarrollarse (Arditti, 1982). La gran diversidad de orquídeas probablemente significa una fuerte diversidad de requerimientos para la germinación de cada especie; de hecho, se han desarrollado varios medios nutritivos para diferentes géneros y especies de orquídeas (Arditti 1967(Arditti , 1982Arditti y Ernest, 1993). ...
... Este investigador además encontró que las semillas de algunos géneros, entre las que se encuentran Cattleya y Epidendrum, germinaban en forma asimbiótica en condiciones in vitro; no obstante se estimó que no siempre es posible desarrollar un medio de cultivo en el cual una determinada orquídea pueda germinar y desarrollarse (Arditti, 1982). La gran diversidad de orquídeas probablemente significa una fuerte diversidad de requerimientos para la germinación de cada especie; de hecho, se han desarrollado varios medios nutritivos para diferentes géneros y especies de orquídeas (Arditti 1967(Arditti , 1982Arditti y Ernest, 1993). ...
... Una vez recolectadas las cápsulas fueron tratadas con etanol a 70% y almacenadas en bolsas plásticas con sílica-gel para su traslado al Laboratorio de Cultivo de Tejidos Vegetales de la Universidad Nacional de Colombia sede Palmira. Los frutos fueron esterilizados en condiciones asépticas según la metodología propuesta por Otero (2004), en la cual, antes de la dehiscencia de la cápsula, esta se desinfecta con hipoclorito de sodio al 1% por 10 min, luego se enjuaga con agua destilada estéril y posteriormente se sumerge en alcohol a 70% durante 5 min (Arditti, 1982); finalmente es flameada superficialmente. ...
... In in vitro culture, carbohydrates are one of the essential supplements as carbon sources for seed germination, seedling growth, micropropagation etc. For the seed germination of orchid, many kinds of carbohydrates (glucose, fructose, sucrose, maltose etc.) have been used as the carbon sources in the culture medium and gave different results in seed germination and subsequent growth (Arditti et al. 1982), indicating that suitable type of carbohydrates differs depending on the orchid species. In Gastrochilus, there have been no reports on the analysis of the effect of carbohydrate type on seed germination and subsequent development of protocorm. ...
... Since Knudson (1922) first reported on orchid (Cattleya) seed germination, glucose, fructose and sucrose had been used for seed germination in many orchid genera as carbon sources alone or in combination (Arditti et al. 1982). Islam and Ichihashi (1999) reported that calli obtained by culturing axillary buds of flower stalk on maltose or sorbitol medium but not sucrose medium initiated protocorm-like body formation and subsequent development into plantlets in Phalaenopsis, Doritaenopsis and Neofinetia. ...
Gastrochilus japonicus (Makino) Schltr. is one of the endangered species in Japan. The aim of this study was to establish an ex-situ conservation method of this species through asymbiotic culture of seeds using media with defined substances without using plant growth regulators. We investigated the influences of different media (New Dogashima, Murashige and Skoog, Modified Knudson C and Modified Vacin and Went), seal materials (surgical tape: not air tight, and Parafilm® M: air tight) for culture vessels, several kinds of carbohydrates (sucrose, maltose, trehalose and glucose), and addition of activated charcoal. The results showed that New Dogashima (ND) medium supplemented with 29.2 mM trehalose was suitable for seed germination (71.9% Parafilm®, 57.5% surgical tape) and subsequent protocorm development. Seal materials for culture vessels with different aeration properties did not affect seed germination. Moreover, ND medium containing 58.4 mM sucrose and 1 g/l activated charcoal was favorable for the plantlet growth after induction of leaves from protocorms. In contrast, ND medium containing trehalose showed slow growth after leaf induction from protocorms among all the carbohydrates tested although it gave the lowest necrosis rate. In seed germination and seedling growth of Gastrochilus japonicus, it might be useful to use media containing trehalose until protocorm formation stage and those containing sucrose and activated charcoal for the stage after leaf induction from protocorms.
... The plant also shows antibacterial activity against Bacillus subtilis and Escherichia coli 16 . There are many reports of its mass propagation through asymbiotic seed and organ culture 17,18,19,20,21,22 . But no information is available on its mycobiont mediated (symbiotic) seed germination. ...
... induced symbiotic seed germination in nearly eighty six percent of the seeds. Earlier workers got 43 -94 percent seed germination of immature seeds of this species on artificial nutrient medium (asymbiotic germination) and reported 55-75% seed germination in majority of cases 17,18,19,20,21,22 . ...
Present study was carried out in four threatened Himalayan orchids. Two of these
[Aerides multiflora Roxb., Rhynchostylis retusa (L.) Blume] were epiphytic and two
[Crepidium acuminatum (D. Don) Szlach., Cypripedium cordigerum D. Don]
terrestrial in habit. The objectives were to isolate and characterize their mycorrhizal
endophytes, to study the efficacy of isolated fungal partners in inducing symbiotic
seed germination in vitro, to study the in vitro asymbiotic seed germination in
chemically defined and undefined media, and to harden the so raised seedlings.
The roots of actively growing plants were used to isolate the fungal endophytes. The
fungal colonization was noticed only in cortical zone of the roots, and the vascular
bundles were always free of fungal infection. In each species, a number of root hairs
were observed protruding out of the epiblema layer, but none of them was found
containing fungal hyphae except in Crepidium acuminatum. The fungal hyphae,
therefore, entered into the root tissue directly through epiblema cells. After reaching
cortex, the fungal mycelium formed loose or sometimes compact networks known as
pelotons. The fungal clumps of adjacent cells were often interconnected by hyphal
threads to repeat the infection. Presently, the fungal endophytes were successfully
isolated from the roots of all species except Cypripedium cordigerium despite
repeated attempts. Morphological and molecular characteristics of various isolates
revealed the identification of two mycobionts (Ceratobasidium sp., Lasiodiplodia
theobromae) in Aerides multiflora, three (Hypocrea sp., Trichoderma sp., Rhizoctonia
sp.) in Crepidium acuminatum, and two (Fusarium sp., Ceratobasidum) in
Rhynchostylis retusa.
For symbiotic germination, seeds from yellowish green and undehisced fruits were
co-cultured with all of the fungal isolates discerned from that species on Oat Meal
Agar medium. Only three isolates i.e. Ceratobasidium sp. (AM301) of Aerides
multiflora, Rhizoctonia sp. (CA103) of Crepidium acuminatum and Ceratobasidum
sp. (RR202) of Rhynchostylis retusa evoked germination and subsequent
morphogenetic changes in their seeds in vitro. As many as 87.00±1.32 percent seeds
germinated in Aerides multiflora, 86.40±1.52 percent in Rhynchostylis retusa and
72.34±1.27 percent in Crepidium acuminatum. The fungal hyphae surrounded the
seeds and penetrated inside mainly from their general body surface; they rarely
entered through embryonic rhizoids/ micropylar end. After entry, the fungus startedix
colonizing the embryonic cells. As embryo size increases, the testa broke to release it
on to the culture medium; the protocorm developed thereafter. Once green protocorms
were formed, they became less dependent on the mycobionts, and were then cultured
on fungus free medium, where they were differentiated further to form healthy
seedlings within 26.40±0.49 wks (Aerides multiflora), 27.83±0.99 wks (Crepidium
acuminatum) and 26.80±0.57 wks (Rhynchostylis retusa).
The fungal requirement of seeds was successfully bypassed in all of the species by
providing an appropriate nutrition in vitro. Three nutritional media were used to
access their ability in provoking seed germination and in bringing about various
morphogenetic changes for subsequent seedling development. The seed germination
percentage was better in case of epiphytic species as compared with the ground
growing ones. Presently, the seeds of C. cordigerum germinated successfully but the
protocorms failed to differentiate leaf and root despite of repeated subculturing. This
may be attributed to their highly complex nutritional requirements. Vitamin enriched
M medium with relatively low calcium and phosphorous contents supported seed
germination in all of the studied epiphytic (Aerides multiflora, highest 78.20±1.64
percent; Rhynchostylis retusa, highest 79.20±0.84 percent) as well as terrestrial
species (Crepidium acuminatum, highest 82.20±2.59 percent; Cypripedium
cordigerum, maximum 61.40±1.67 percent). MS medium with relatively high
nitrogen contents also gave positive response for seed germination in all of the studied
orchid species (Aerides multiflora with highest 83.20±0.84 percent, Rhynchostylis
retusa with highest 79.20±5.54 percent, Crepidium acuminatum with highest
76.40±2.30 percent and Cypripedium cordigerum with highest 81.20±1.30 percent).
MS medium is highly enriched with macro and micro elements, and usually results in
high germination of seeds and better development of seedlings thereafter. The
chemically undefined PDA medium supported comparatively lower seed germination
in all the studied orchids. It was successfully employed for seedlings development in
A. multiflora and R. retusa. However, in Cripedium acuminatum, the protocorms
failed to differentiate further, while in Cypripedium cordigerum no development was
observed after spherule stage despite of repeated subculturing. As terrestrial orchids
are nutritionally complex, chemically defined media are generally better for their
germination, growth and development. Activated Charcoal (AC; 2gl-1) was used in all
culture media for accessing its effect on seed germination and further morphogenetic
changes. It not only improved germination percentage but also proved effective inx
reducing time period between seed sowing and seedling development in majority of
the nutritional combinations.
The effect of auxins (IAA, NAA) and cytokinins (BA, KN) was tested individually
and in combination on germination and seedlings development. IAA (Indole-3-acetic
acid) at a concentration of 0.5mgl-1 proved to be the best for early protocorm and
seedling development in Crepidium acuminatum and Rhynchostylis retusa. NAA (α-
Nepthlene acetic acid) also had a positive effect of on seed germination, proliferation
and differentiation. BA (6-Benzylaminopurine) at lower concentration accelerated the
seedling growth and development in majority of cases. At the concentration of
1.0mgl-1, it proved better for early development of protocorms and early
differentiation of leaf in Aerides multiflora. Kinetin (6-furfurylaminopurine) was
found beneficial for early development of protocorms in Rhynchostylis retusa and
Cypripedium cordigerum. A combined treatment of auxins and cytokinins supported
better germination, proliferation and healthy development of seedlings in all of the
presently investigated species.
Hardening (acclimatization) is important because the seedlings produced in vitro
under comparatively higher humidity conditions are much susceptible to desiccation
when transferred to the greenhouse. Symbiotically raised seedlings showed better
survival success when transferred to the ex vitro conditions, and their mortality rate
was very less. Such seedlings exhibited higher survival rate during lab to land transfer because of the presence of mycorrhizal endophytes in their roots that help in supplying the requisite nutrients in the newer environment.
... The seeds were spread on the surface of the sterile medium and incubated for 4 weeks in a controlled growth room at 26 ± 1 • C and a 16 h/8 h light/dark photoperiod with a light intensity of 30 mmol·m −2 ·s −1 provided by a Phillips ® cool-white, fluorescent lamp. Visible protocorms of stage three [17] were observed at the end of the culture period. ...
... NAA was found to be more superior than other PGRs for the PLBs' induction, such as Aranda [31], Dendrobium candidum [25], and Dendrobium aqueum [32]. NAA is also frequently used in combination with cytokinins, similar to BAP in other orchid species [17,33,34]. However, our results demonstrated a better regeneration using BAP, although the differences between NAA and BAP were insignificant. ...
This first-attempt study used microtome-based methods to generate a thin cell layer culture for the micropropagation of Phal. Hwafeng Redjewel × Phal. New Cinderella. Protocorms were embedded in various agarose concentrations (8–12%, w/v) and dried from 1 to 8 h before sectioning with a microtome. Optimal conditions for slicing sections of 100 to 300 μm were achieved when the protocorms were embedded at 10% (w/v) agarose and dried for 4 h under laminar flow, and the hardness of the agarose block under these conditions reached 641.8 ± 9.5 g·cm−2. The sectioned protocorms that were cultured on an MS medium supplemented with 1.2 mg·L−1 6-benzylaminopurine and 0.1 mg·L−1 α-naphthaleneacetic acid were capable of growth and differentiated through the neoformation of protocorm-like bodies (PLBs) and/or callus before subsequent regeneration into plantlets and development into healthy plants in a nursery environment. The results of this study demonstrate that microtome-based tTCL is a reliable and promising approach for mass propagation and possible virus-free propagation objectives for Phalaenopsis.
... The number of genera magnificent flower to found in entire orchid family. This varies from 500-800 and the number of species from 15000has contributed much to the work of hybridists producing 35000 according to various repots (Anonymous, 2008;Arditti, 1979;Arditti et al.,1982;Kotia et al., 2010;Aktar et al., 2008). However, some flowers for cut flower market. ...
... This might be attributed to the inhibitory influence of nitrogen in the form of ammonium sulphate in B5 medium or mixtures of vitamins present in both B5 and NN media on seedling growth (Parthibhan et al., 2012;Pyati et al., 2002;. The poor response in terms of seed germination and growth of the protocorms in KC medium could have been due to the lower amount of nutrients and vitamins present in KC medium which were not sufficient for complete development of the seedlings (Arditti et al., 1982). However, Nongrum et al. reported enhanced seed germination of Coelogynae ovalis and C. nitida in KC medium. ...
An efficient protocol for seed germination and micropropagation of Cymbidium ensifolium (L.) Swartz. was established. Four nutrient media were used for seed germination and early protocorm development: Murashige and Skoog (MS), half –strength MS, Knudson ‘C’ (KC), and Vasin and Went (VW); combinations and alone of four plant growth regulators i.e. 6-benzylaminopurine (BAP), kinetin (KN), α-napthalene acetic acid (NAA), and indole-3-butyric acid (IBA) were studied. MS medium was found as most ideal for seed germination (98±0.48) and lowest in VW (71.12 ±0.42). 3 months old protocorm were sub cultured on fresh MS medium supplemented with different concentrations of BAP, KN, NAA, and IBA alone and in combination. After 30 days highest secondary protocorms (21.25±0.63) were observed in MS medium containing BAP (4.0μM). MS medium supplemented with 8μM IBA induced the maximum roots per shoot. After 16 days of transfer to green house the survival rate was 88%.
... The choice of culture medium strongly affected germination, presumably because of differences in the balance and supply of organic and inorganic nutrients (Arditti, 1982;Arditti and Ernst, 1984;Zeigler et al, 1985;Van Waes and Debergh, 1986). According to Yam et al (1989), the nutritional requirements of germinating orchid seeds vary due to their physiological state and this may be species specific. ...
Aeridas rosea and Eria lasiopetala are the species of rare and endangered orchids of uttrakhand region. These species deal with the in vitro propagation methods. For green pod culture, three Nutrient media Murashige and skoog (MS Media), Knudson Media (KC Media) and stock Solution of MS Media were tested by adding different concentration of growth regulators. MS Media supplemented with growth regulators 1.5mg/L NAA and 1.5mg/L BAP were found to be the most effective media for the development of protocorn like body(PLBs). This study is helpful in the propagation of these rare Orchids Plants at lower elevation, which could be a successful effort of conservation of rare Orchid plants and this could also reduce pressure on its population in the Wild. How to cite : Richa Chauhan, Ashutosh Mishra and Anuradha Saini (2024) Effect of media on regeneration of rare orchid species through tissue culture method. Biochem. Cell. Arch. 24, 981-984.
... The plantlets developed through embryo culture or seed culture is generally subjected to micropropagation for rapid multiplication. This technique is helpful for commercialization of orchids.Seed culture technique and micropropagation is taken into consideration of this study.The seed culture technique helps in germination of plants where the normal growth is difficult.This technique ensures much better germination particularly in the genera where germination is not possible due to some abnormality within the seeds(Arditti et al., 1982;Linden, 1980;McIntyre et al., 1972;Pathak, 1989; Pathak et al., 1992; Vij and Pathak, 1988; Vij et al., 1995; Yam and Weatherhead, 1988). The orchid seeds are minute and exhibit very poor level of differentiation. ...
Bellamya bengalensis (Lamarck, 1822) in one of the most important fresh water, edible, widely distributed snails found in India. This species is present throughout Asia and Africa, and of great medicinal as well as socioeconomic values. Moreover, it is a cheap source of protein which is even higher than some common fish and red meat groups. Besides human, it is also a beneficial food source of many aquatic fishes and birds; hence, Bellamya bengalensis is one the significant part of aquatic ecosystem. Studies revealed that it has been used in the treatment of different human diseases like chronic gastric disorder, arthritis, joint pain, rheumatism, cardiovascular disease, night blindness, asthma, rickets, diarrhea etc. Although it possesses high protein content, it is being neglected than other proteinaceous resources in terms of food choice. Various bacteria present in the gut and flesh of Bellamya bengalensis act positively in the growth and survival of the snails. As snails are mostly found in the polluted water bodies, toxic elements present in polluted water affects the host-bacteria equilibrium, and hence changes in the metabolism and physiology of snails may have been observed. Acute and chronic toxicity studies with different toxic elements like formic acid, copper sulphate, fluoride, mercury etc. from industrial effluent showed behavioral and respiratory alterations along with decreased oxygen consumption rate in Bellamya bengalensis. This review provides an overview of ecological importance and its application on Bellamya bengalensis. Further studies will be done on physiological alterations upon application of toxic elements.
... The plantlets developed through embryo culture or seed culture is generally subjected to micropropagation for rapid multiplication. This technique is helpful for commercialization of orchids.Seed culture technique and micropropagation is taken into consideration of this study.The seed culture technique helps in germination of plants where the normal growth is difficult.This technique ensures much better germination particularly in the genera where germination is not possible due to some abnormality within the seeds(Arditti et al., 1982;Linden, 1980;McIntyre et al., 1972;Pathak, 1989; Pathak et al., 1992; Vij and Pathak, 1988; Vij et al., 1995; Yam and Weatherhead, 1988). The orchid seeds are minute and exhibit very poor level of differentiation. ...
Subhas Sarobar is a medium-sized artificial lake situated in the northeastern region of Kolkata, West Bengal which is controlled by the Calcutta Improvement Trust. This body of water is home to a variety of organisms, including bacteria, benthos, nekton, phytoplankton, and zooplankton, all of which contribute to a healthy and interdependent ecosystem. Seasonal variations in temperature, precipitation, and humidity have an impact on the physicochemical characteristics of the water, which could lead to a shift in the faunal population. Additionally, residential effluents are used on this aquatic system. Therefore, in addition to seasonal variations, home spills, washing clothes and utensils, taking baths, and disposing of plastic garbage next to the lake water all affect the physico-chemical characteristics of the water. The lake water quality ultimately declines as a result of all these human activities. The combination of high nutrient content and low DO creates an ideal habitat for the growth of bacteria in the lake system, including pathogenic bacteria that might potentially harm public health as well as other aquatic creatures. Pathogenic bacteria species found in water are dangerous and can cause several illnesses to human beings. It is possible to successfully stop the spread of disease(s) if the traits and specifics of pathogenic bacteria are understood.
... The ripeness of the orchid fruits will support of the success of the seed germination process. Capsule maturity one of several factors that affect of success of in vitro germination of orchid seeds [9]. ...
... The nature and quantity of growth hormones have significant effects on the germination of orchid seeds (Arditti, 1979). Immature seeds of orchid species from northern temperate regions are more difficult to germinate than mature seeds (Arditti et al., 1982). In this study, the MS basal medium is nevertheless found to be effective for the in vitro seed germination. ...
... Seed production is high in orchids, with each capsule containing thousands or millions of small seeds, depending on the species (Arditti, 1967;Arditti & Ghani, 2000). Germination is generally difficult in temperate terrestrial orchids (Arditti et al., 1982Rasmussen, 1995Miyoshi & Mii, 1998), with only 0.2-0.3% of the millions of seeds in a capsule germinating in nature (Citil & Tekinşen, 2011). Since the tiny seeds have a small embryo with no endosperm, they require a mycorrhizal relationship for germination and development in nature, receiving water, nitrogen, carbohydrates, vitamins and organic compounds via mycobionts (Kauth et al., 2008). ...
Aim of study: Despite protection by international agreements, millions of orchid tubers are harvested from their natural distribution areas each year. Of these species, Himantoglossum robertianum is locally threatened due to overharvesting and requires precautionary measures to ensure its protection. Reproduction of the species in an asymbiotic environment is imperative for providing ex-situ protection. There are no studies on optimum germination temperature in H. robertianum. This study aimed to germinate H. robertianum seeds in-vitro under asymbiotic conditions. Area of study: The study was carried out at the Silviculture Laboratories of Bursa Technical University, Faculty of Forestry, Department of Forestry Engineering. Material and methods: H. robertianum seeds were used in the study. Seeds were germinated in five replications at four different temperatures (10, 15, 20, and 25°C (± 0.5°C). The study was conducted for 275 days under dark conditions with Sıgma-Phytamax P-6668 used as the medium. Main results: The highest germination was 23.8% at 20°C and germination was not obtained at 10°C. While germination was faster at 25°C in the first 18 weeks, germination accelerated at 20°C after 18 weeks. Highlights: These results indicate that temperature is an important factor in the germination of H. robertainum seeds.
... Consequently, in the natural environment, the seed embryo may imbibe but fail to develop further unless an exogenous supply of carbohydrate is provided or mycorrhizal association occurs (Smith and Read, 1997;Chugh et al., 2009). In the laboratory, orchid seed germination is possible in vitro on a medium that varies in complexity depending on the species but generally includes macro-and micro-elements and a source of carbon (Arditti et al., 1982;Deb and Pongener, 2011). Norstog medium, with amino acids, may be effective with epiphytic species, while Knudson C, with activated charcoal, can support the germination of tropical terrestrial species (Nadarajan et al., 2011). ...
Generally, orchids produce dust-like seeds in which endosperm reduction and embryo undifferentiation represent a derived state shared with species in about 11 other plant families. Orchid seeds are proposed to have a special kind of morphological or morphophysiological dormancy. We test this proposition, overcoming several design limitations of earlier studies, specifically that the in vitro germination method for orchid seeds uses pro-oxidants for disinfection and incorporates nitrate in the medium; both ‘treatments’ might contribute to dormancy breaking, potentially confounding judgement on the depth and nature of the dormant state. Seeds of the tropical orchid Dendrobium cruentum Rchb. f., were sown both in vitro , on a nutrient medium, and ex vitro , on plain agar omitting prior disinfection with sodium hypochlorite. Seeds previously stored and fresh seeds were incubated under combinations of vitro conditions, light treatments, constant or alternating temperatures and nitrate concentration. Seeds of D. cruentum are very small but have a large embryo that occupies most of the seed. Over a range of constant temperature seeds germinated to the spherical protocorm stage just as well ex vitro as in vitro . Neither light nor nitrate were prerequisites for ex vitro germination. The ability of D. cruentum seed to germinate in the absence of environmental or chemical stimuli suggests that mature seed can be non-dormant. Our results support the proposition that neither all DUST seed fit a dormancy class nor all orchids produce morphological or morphophysiological seeds. Finally, embryo/seed volume determinations in orchids may prove as valuable in studies on the evolution and ecology of germination and dormancy as embryo:seed ratios in other angiosperm species.
... Widely used chemicals for sterilisation include solutions of various hypochlorites, Na 2 salt of dichloroisocyanuric acid, hydrogen peroxide, mercuric chloride etc., although their concentration and time of exposure might vary from species to species. Additionally, some workers preferred to pre-soak the exposed orchid seeds in a sugar-based solution like honey before adding the sterilising agent to increase the efficiency of the process (Arditti et al., 1982;Snow, 1985). It was opined that sugar or honey solution induced the bacterial or fungal spores to germinate thereby making them more vulnerable as disinfecting agents which otherwise remained hidden inside the porous seed surface. ...
Asymbiotic seed germination has revolutionised the commercial production of exotic orchids as well as conservation of rare and endemic ones. However, the success of the process largely depends on the sterilisation procedure adopted to disinfect the minute dust like orchid seeds. The present study incorporates the technique of centrifugation method to successfully disinfect the exposed seeds obtained from the dehisced capsule of Phaius tankervilleae, which is an ornamental orchid endemic to Northeast India. The seeds of P. tankervilleae showed 100% disinfection as well as germination efficiency (with 5% NaOCl and 8 mins exposure) with no risk of physical damage to the seeds due to the centrifugation process (4 mins at 4000 rpm under 4˚C). Inoculated seeds showed the first sign of swollen embryos (PLBs) after 5 weeks of culture in Murashige and Skoog (1962) (MS) medium. PLBs obtained were subcultured on MS medium supplemented with different plant growth regulators for healthy proliferation and shoot multiplication. MS medium supplemented with 2 mg/L each of BAP and KN resulted in best overall shoot and root development. Therefore, the present study reports a simple yet effective sterilisation procedure to disinfect exposed P. tankervilleae seeds which is quick, easy and very efficient for mass scale propagation as well as ex-situ conservation of this valuable orchid species and other orchid species as well.
... Many cold-hardy terrestrial orchids exhibit low seed germination, which is often attributed to low viability or dormancy. Immature seeds of many orchid species have been shown to germinate more readily than mature seeds [20][21][22]. Mature seed mentioned in orchids actually refer to the seeds from dehiscent capsules. Several factors may contribute to the increased germinability of immature seeds. ...
Two prevalent issues with Gastrodia elata growing in a natural wild environment are the necessary symbiotic seed germination with Mycena spp. and the long growing cycle of protocorms into mature rhizomes with Armillaria mellea. This study explored the most suitable nutrients to germinate seeds and the developmental stages and propagated protocorm/juvenile rhizomes (jrhs) through a tissue culture approach. Adding humic acid, mashed potato, peptone, and (indole-3-acetic acid) IAA to the 2 g/L Hyponex 7-6-19 basal medium accelerated seed germination into protocorms with an average length of 2 mm. The nutrients best for the in vitro propagation of jrhs consisted of 1 g/L Hyponex 7-6-19 and MS salts with 1/2 NH4NO3 and KNO3, 2 mg/L BA, 2 mg/L NAA, 2 g/L tryptone, 2 g/L glutamic acid, 1 g/L chitosan, 10 g/L apple puree, 20 g/L mashed potato, 100 mL/L coconut water, and 1.5 g/L A. mellea powder, at a pH level of 6. The maximum length and number of jrhs were 17.2 ± 0.8 mm and 76.6, respectively, in two subcultures. The in vitro propagation system started from seeds of G. elata can be employed as a potential method to propagate protocorm/jrhs for the cultivation of mature rhizomes from indoor or outdoor cultures.
... The culture media with organic supplements showed an increase in germination, presumably because of differences in the balance and supply of organic and inorganic constituents [17] [18] [19] [20]. The greater germination percentage, when compared with the control, was found in culture media that contained coconut water (CW) alone. ...
... D. macraei Lindl.: The whole plant is useful for treating bronchitis, throat problem, fever, asthma, and as an aphrodisiac [68]. It can also be utilized as a tonic for general debility [69]. ...
... (Nhut et al. 2005); PLB in C. Sleeping Nymph, Cym. giganteum (Vendrame and Khoddamzadeh 2016;Vyas et al. 2010;Hossain 2011), etc. Seeds, shoot tip, and inflorescence bud are popular used for initial culture of Cymbidium (Arditti 1982;Choi et al. 1998;Paek and Murthy 2013). Cymbidium forrestii was clonally propagated using seed-derived rhizomes as explants (Paek and Yeung 1991). ...
With over a millennium history of cultivation, Cymbidium species and hybrids is one of the most commercially important orchids in world floriculture industry. The Cymbidium genus has near 75 species with ornamental and medicinal importance and thousands of hybrids used for pot and cut flower production. Plant tissue culture techniques for breeding and large-scale propagation associated with greenhouses cultivation in sophisticated systems were used to accelerate the plant development until commercial flowering and aiming the high quality, uniformity and productivity of flowers. In this review, the overview picture in Cymbidium has been deeply discussed, such as diversity and ecology of species, karyotype characterization, identification and characterization of species and cultivars by molecular markers, aroma compounds biosynthesized in some species, micropropagation of species, somaclonal variation originated from clonal propagation and applications in breeding, production of biomass and bioactive compounds, omics approaches and symbioses of some species with a mycorrhizal fungus. The information presented here is useful to understand actual application of biochemical, biotechnological, cellular and molecular tools associated with ecology, breeding and propagation in Cymbidium species and cultivars.
... in late capsule group than in early capsule group ( Figure 1). This finding contradicts with the earlier observations for orchid seed germination as lower germination frequencies have been achieved by culturing mature seeds than immature seeds (Arditti et al., 1982;Rasmussen, 1995), since on maturity, integuments become impermeable to water (Kauth et al., 2008). However, to another view, sparse cuticular deposition in mature orchid seeds can make testa less hydrophobic resulting in greater germination of mature seeds (Hsu & Lee, 2012). ...
... They must be in a symbiotic relationship with fungi for germination (Veyret, 1974;Maheshwari, 2005;Baskin and Baskin, 2014). The epiphytic species germinate better than the terrestrial ones due probably to their simpler nutritional requirements (Arditti et al., 1982;Rasmussen, 1995;Miyoshi and Mii, 1998). ...
Although orchids are the most rich family of the plant kingdom, they make up the most destroyed group. Due to human impact, environmental factors, tourism and settlement, ornamental plants or food collection, the spread areas are getting narrower and endangered every day. Two of these endangered species due to their use in the food industry are Himantoglossum robertianum (Loisel.) P. Delforge and Orchis italica Poiret. Both of these sahlep orchids taxa are preferred by collectors in terms of their tuber size and the glucomannan contains and are heavily destroyed. Excessive destruction brings some pollination and fertilization problems. Orchid seeds do not have an endosperm and need a symbiotic relationship to germinate. To germinate in an asymbiotic environment, it is necessary to apply the seeds that break dormancy and keep them in a special medium and environment. The structure of the seed coat is important in eliminating the germination barrier in asymbiotic production. In this study tuber and seed morphological and physiological properties of both species were determined. Morphological features such as plant height, tuber weight and dimensions, seed sizes, seed weight, the number of seeds in 1 g, and physiological features such as seed moisture, shell structure and fullness of seeds were determined. The causes of empty seed formation are interpreted in light of the literature.
... Keywords: Cypripedium, Pindari valley, Kumaun West Himalaya, Conservation. * Vij et al., 2013;** Arditti et al., 1982;Arditti et al.,1984 along with their habitat characteristics, medicinal uses and status are given in Table 1. ...
The present study is an attempt to assess distribution pattern and specific habitat requirements of three threatened species of genus Cypripedium i.e. C. cordigerum, C. elegans and C. himalaicum in Pindari valley, Kumaun West Himalaya. Results revealed that partially shaded places and humus-rich soil form the ideal habitat conditions for the growth of this species. However, restricted distribution and low plant density in the study sites reflect the critically rare status of this species. The species is declining in natural habitat due to grazing, habitat loss, and unsustainable collection. Hence, conservation planning is needed to protect the species and its habitat.
... and Laelia Lindl. species (Arditti et al. 1982;Damon et al. 2004). However, the establishment of protocols for in vitro asymbiotic germination of orchid seeds is species-specific and depends on several factors such as; capsule maturity, components of culture media, light and temperature (Arditti 1992). ...
Orchids require mycorrhizal fungi (OMF) for their germination and growth. Propagation and re-introduction initiatives would likely require inoculation with such fungi. All Chilean Orchidaceae species are terrestrial and likely associate with OMF. We collected adult individuals of the endemic Chilean orchid Chloraea gavilu and transported them to a glasshouse where we obtained mature capsules through manual auto-pollination. We asymbiotically germinated seeds in vitro using Malmgren Modified Terrestrial Orchid-Medium (MM). Embryos were put in glass flasks with MM where roots and leaves developed for 16 weeks. Plants were then transplanted to 165 mL pots and randomly separated into three groups; plants inoculated separately with Ceratobasidium OMFs isolated from two Chilean orchid species (Chloraea virescens and Codonorchis lessonii), and uninoculated (control) plants. Plants were then put in a growth chamber. Three months later, inoculated individuals showed pelotons inside par-enchyma cells in the roots. Four months after inoculation, mycorrhizal plants had higher root and shoot biomass compared to control plants. At the end of the experiment, the highest mycorrhization was achieved with the Ceratobasidium strain isolated from C. lessonii. The artificial mycorrhization of endemic orchids could be a key strategy for future conservation and propagation initiatives, especially for endangered or commercially interesting species. ARTICLE HISTORY
... In vitro germination of seeds is an important part in the orchid multiplication and conservation programmes since the 'dust seeds' are tiny and contain few food reserves. Knudson 3 demonstrated the possibility of by-passing the fungal requirement of orchid seeds during in vitro germination and since then non-symbiotic seed germination has been accepted as an important tool for propagating orchids 4 . The non-symbiotic seed germination potential of fertilized ovules has been positively tested in several commercially viable and/or threatened orchid taxa 5,6 . ...
Cleisostoma racemiferum, an epiphytic orchid of primary forest under threat in their natural habitat, was studied. Immature seeds of different developmental stages [(8-20 week after pollination (WAP)] were cultured on Knudson 'C', Mitra et al and MS media supplemented with sucrose (0-3%, w/v), coconut water (CW, 0-20%, v/v) and NAA (0-30 μM) + BA (0-8 μM) singly or in combination. After 7 week of culture, first sign of germination was recorded as nodular swelling of seeds. Amongst the three different basal media tested, better germination was supported by MS medium, followed by Mitra et al and Knudson 'C' media containing sucrose (3%) and NAA (10.0 μM) + BA (8.0 μM). Of various developmental stages of the seed, better germination was obtained from green pods of 16 week after pollination. Younger seeds did not show any sign of germination, while mature seeds exhibited delayed and deformed germination. Though incorporation of CW in the medium did not show much influence on seed germination, but 15% (v/v) CW in the initiation medium enhanced the early differentiation of protocorm-like bodies (PLBs) into plantlets. Within 14-16 week of culture on germination medium, the PLBs started releasing the first set of leaflets. The advanced stage PLBs were converted into rooted plantlets on MS medium containing IAA (10.0 μM) + Kn (9.0 μM). Although the medium containing NAA (10.0 μM) + BA (8.0 μM) resulted in multiple shoot buds, but the leaves were thin and etiolated. Further, the medium enriched with NAA (10.0 μM) + Kn (9.0 μM) resulted in stunted growth of plantlets, while presence of IAA (10.0 μM) + BA (8.0 μM) resulted in plantlets with poor roots.
... Multiplication of orchids through in vitro seed germination technique have been developed for several species and are most commonly used method for multiplying different orchid species and hybrids representing diverse habits and habitats. 3,49 Under culture conditions germination of seeds are affected by several physicochemical factors and it is requisite to optimize seed germination for each species of orchid. A number of media such as Murashige and Skoog, 43 Mitra et al., 41 Kundson 33 and Gamborg et al., 21 etc. have been used for seed germination and propagation through tissue culture. ...
Orchids (Family Orchidaceae) are ornamentally valuable and unique group of flowering plants. They are valued mostly for their long lasting and diverse morphology of flowers. Some orchids are highly prized due to their application in food, flavor and medicinal industry. Seed germination and vegetative propagation are the methods through which
orchid plants regenerate in nature. Both these methods of regeneration are quite slow to fulfill orchid demand. Plant tissue culture technique has been used extensively in multiplication of orchids. Although seed germination produces heterogenous population but most of orchid species are multiplied by asymbiotic seed germination. Propagation
through protocorm-like-bodies (PLBs) or direct from explant regeneration is quite more advantageous to multiply orchids. Artificial seed technology is another method used for orchid multiplication, transport and conservation of endangered orchid species. Culture of medicinal orchids for production of bioactive metabolites could be used as
substitute for dependence on natural population.
... Here, it was also found that addition of coconut water was the most beneficial, but Knudson medium had a non-significant higher germination rate than modified MS. KC medium was first developed to increase germination success of orchids seeds (Curtis and Spoerl 1948) and often used for tropical epiphytic orchids (Arditti 1982). KC medium has little macro-and micro-salts and no vitamins. ...
Asymbiotic seed germination of Thunia alba (Lindley) H.G. Reichenbach was tested on different media in order to propagate and preserve its genetic diversity. In addition different plant hormones were used in the similar germination experiments. Results from these experiments revealed that Knudson medium (KC) had the best germination ability but Mitra and modified Murashige and Skoog (MS) also showed good responses for growth and root formation in the seedling after eight weeks. Coconut water (CW) was the most efficient for promoting germination and growth, addition of 0.046 µM and 0.46 µM Kn also induced better germination rate and formation of leaves. The seedlings after eight months were transferred and acclimatized to grow in the garden on a medium containing bark and crushed concrete blocks. Although the species is amongst the rare orchid species that can successfully propagate asexually in the wild it is demanding for substrate and climatic conditions.
... D. macraei Lindl.: The whole plant is useful for treating bronchitis, throat problem, fever, asthma, and as an aphrodisiac [68]. It can also be utilized as a tonic for general debility [69]. ...
Plants belonging to the Dendrobium genus occupy a dominant position among the orchids because of their high ornamental and therapeutic values. They are widely popular in the international floriculture trade as they bear stunning flowers with diverse coloration, varied forms, and patterns. The ethnomedicinal uses of these orchids are also prominently found due to the possession of immense medicinal properties. Excessive exploitation through rampant unregulated collection and widespread habitat destruction have dwindled the Dendrobium natural populations at an alarming rate. The fast and reliable micropropagation techniques offer an alternative to the slow conventional methods of Dendrobium propagation. The rapid in vitro regeneration of genetically stable Dendrobiums is essential for effective germplasm conservation and large-scale orchid commercialization. Several genetic stable Dendrobiums have been successfully propagated by ascertaining the clonal fidelity of the regenerants using different molecular markers. This chapter focuses on the uses of Dendrobiums as important ethnomedicine, their in vitro propagation, and clonal assessment for producing genetically stable orchids using DNA markers.
... The fungal requirement can be bypassed with incorporation of sugars along with adjuvant in vitro. Since Knudson reported the possibility to bypass the fungal requirement for in vitro orchid seeds germination, asymbiotic germination has emerged as an important tool for orchid propagation [1,2,6,17]. ...
Paphiopedilum insigne (Orchidaceae) is a vulnerable commercially important orchid owing to its attractive flowers. A successful attempt was made to develop an efficient low cost in vitro propagation protocol from immature embryos. Immature embryos from the 210 days aged green pods was cultured on nutrient media conjunct with organic carbon, plant growth regulators under different light conditions. Within 38 days first sign of germination was recorded as nodular swelling followed by protocorm-like bodies (PLBs) formation in 85% of cultured seeds after 70 days of culture on MS medium supplemented with sucrose (3%), NAA and BA (2 and 6 µM in combination). The PLBs differentiated to plantlets and culture proliferated on MS medium + sucrose (3%) + 4 µM each of NAA and BA where 9 shoot buds developed per explants. Well rooted plantlets were primary hardened by maintaining on 1/10 th MS liquid medium and mixture of low cost substrata (sand: Decaying organic matter: brick pieces: Charcoal pieces: dried cow dung at 1:1:1:1:1 ratio. Hardened regenerates were transplanted to community pots along with hardening substrata and maintained under semi-controlled condition in poly-house where 75% transplants survived. Over 1500 regenerates are established in the wild.
... Orchid requires a combination of multiplicity of factor for continued reproduction in nature. The propagation of this species through sexual means is a very slow process as its seeds lack endosperm and need fungal stimulant for germination in nature; the fungus is believed to augment the carbohydrate, auxin and vitamin transport in the orchid (Arditti et al., 1982). In nature, only 2 to 5% of seeds germinate (Rao, 1997) even if they do so, the seeds take a long time for their germination and any disturbance in the habitat or physical environment destroys the whole population. ...
Dendrobium primulinum Lindl. (D. Primulinum L.) is one of the important epiphytic orchid species for horticultural and commercial use. It is listed as a rare and critically endangered species of orchid. The present study is intended to conserve the orchid species through the micropropagation technique. The in vitro seed germination and seedling growth was carried out by taking a mature pod of D. Primulinum L. in Murashige and Skoog (MS) media, and the MS media supplemented with a varied concentration (0.5, 1.0, 1.5 and 2 mg/L) of 6-benzylaminopurine (BAP), and a fixed concentration (0.5 mg/L) of α-naphthalene acetic acid (NAA). Sequential phases of seed germination, protocorm formation, and seedling development in the presence of growth regulators were determined in the study. The significance of hormonal effects was determined by using One-Way Analysis of Variance (ANOVA, p≤0.05). The seed germination started after two weeks of culture in the media supplemented with BAP. The maximum seedling growth was obtained in the media supplemented with 1.5 mg/L of BAP. Although the hormone-free basal medium revealed an ideal condition for seed germination and spherules formation, the presence of an appropriate concentration of growth regulators such as 0.5 mg/L NAA or a combined 0.5 mg/L BAP + 0.5 mg/L NAA expressed a synergistic effect to enhance the protocorm formation and seedling development.
Key words: In vitro, Dendrobium primulinum, 6-benzylaminopurine (BAP), α-Naphthalene acetic acid (NAA).
... Plant tissue culture is the only powerful technique for the multiplication and conservation of orchid plants. [10,11,12] In our study, we focused on developing an efficient protocol for the large-scale multiplication of important medicinal orchid V. roxburghii through in vitro seed germination and indirect regeneration from leaf explants. Further, an attempt was made to reduce the cost of in vitro micropropagation of V. roxburghii orchid through novel acclimatization technology. ...
... Photoperiod 24/0 h L/D also played an important role in seed germination only in MM media. Generally the light induces seed dormancy in terrestrial orchid seeds (ARDITTI et al., 1981;ERNST, 1982; VAN WAES & DEBERGH, 1986;YAMAZAKI & MIYOSHI, 2006) but in the present investigation, it was observed that light also enhances the seed germination, similar observations are reported by ZETTLER et al., (1994) in an endangered orchid, Platanther aintegrilabia and RASMUSSEN et al., (1990) in Dactylorhiza majalis. ...
Anthropogenic impact on specific habitats results to the destruction of many key species of ecosystem. Terrestrial orchids grow in such specific undisturbed habitats and this makes the species rarer and prone to threat. Similarly Peristylus lawii, a terrestrial orchid grows in specific habitat at the base of the hills in shady places. Due to overgrazing and habitat destruction the population was confined to such places where grazing animals does not reach. In present investigation, six different media (Vacin and Went, Malmgren Modified Terrestrial Orchid Medium, Modified Knudson C, ½ Murashige and Skoog, Lindemann and Van Waes and Debergh (BM1) were used to identify the nutritional need of Peristylus lawii. The effect of photoperiod was analyzed for asymbiotic seed germination (0/24, 16/8, 24/0 h L/D) and in vitro seedling development (8/16, 12/12, 16/8 h L/D). Seed germination percentage was the highest on MM and BM1 after 18 weeks culture (MM-90.1%) (BM1 76.66%) on 0/24 h L/D and 16/8 h L/D respectively. In vitro seedlings cultured under 16/8 h L/D conditions produced more height of the seedling (5.3cm), leaf length (2.1cm) and tuber length (3.43cm) and tuber diameter (0.16 cm). Tuber developed on higher concentrations of BA produced more tuber length (4mg/l-4.1cm, 5mg/l-4.5cm) and more fresh weight (673mg & 831mg respectively).
... Photoperiod 24/0 h L/D also played an important role in seed germination only in MM media. Generally the light induces seed dormancy in terrestrial orchid seeds (ARDITTI et al., 1981;ERNST, 1982; VAN WAES & DEBERGH, 1986;YAMAZAKI & MIYOSHI, 2006) but in the present investigation, it was observed that light also enhances the seed germination, similar observations are reported by ZETTLER et al., (1994) in an endangered orchid, Platanther aintegrilabia and RASMUSSEN et al., (1990) in Dactylorhiza majalis. ...
Anthropogenic impact on specific habitats results to the destruction of many key species of ecosystem. Terrestrial orchids grow in such specific undisturbed habitats and this makes the species rarer and prone to threat. Similarly Peristylus lawii, a terrestrial orchid grows in specific habitat at the base of the hills in shady places. Due to overgrazing and habitat destruction the population was confined to such places where grazing animals does not reach. In present investigation, six different media (Vacin and Went, Malmgren Modified Terrestrial Orchid Medium, Modified Knudson C, ½ Murashige and Skoog, Lindemann and Van Waes and Debergh (BM1) were used to identify the nutritional need of Peristylus lawii. The effect of photoperiod was analyzed for asymbiotic seed germination (0/24, 16/8, 24/0 h L/D) and in vitro seedling development (8/16, 12/12, 16/8 h L/D). Seed germination percentage was the highest on MM and BM1 after 18 weeks culture (MM-90.1%) (BM1 76.66%) on 0/24 h L/D and 16/8 h L/D respectively. In vitro seedlings cultured under 16/8 h L/D conditions produced more height of the seedling (5.3cm), leaf length (2.1cm) and tuber length (3.43cm) and tuber diameter (0.16 cm). Tuber developed on higher concentrations of BA produced more tuber length (4mg/l-4.1cm, 5mg/l-4.5cm) and more fresh weight (673mg & 831mg respectively).
... It is a popular ornamental orchid used as a potted plant or garden plant (Moir, 1983). In general, seeds of terrestrial orchids are considered more difficult to germinate than those of epiphytic orchids (Arditti et al., 1982). The reasons for the poor germination may relate to the impermeable seed coat (Veyret, 1969;Lee et al., 2005) and/or the accumulation in mature seeds of inhibitory substances, such as phenolics (Kako, 1976) and abscisic acid (ABA) (Van Waes and Debergh, 1986;Van der Kinderen, 1987). ...
The phytohormone abscisic acid (ABA) is involved in regulating seed dormancy and germination. A crucial step of ABA biosynthesis in higher plants is the oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED). Seed development in orchids is unusual because the embryos are minute in size, without obvious histodifferentiation, and lack endosperm. To understand the regulation of ABA biosynthesis in orchid seeds, we isolated and characterized a full-length cDNA encoding an NCED homolog, PtNCED1, from developing seeds of an ornamental orchid, Phaius tankervilliae. Germination percentage was high at 90 days after pollination (DAP), when a globular embryo proper with a degenerating suspensor was evident. After 90 DAP, seed maturation was accompanied by a decrease in water content and a concomitant increase in ABA content and PtNCED1 mRNA level along with a marked decrease in germination percentage. Mature seeds pretreated with NaOCl solution lowered ABA content and improved seed germination. Moreover, after seed germination, developing protocorms could respond to dehydration stress. Dehydration of protocorms stimulated an increase in PtNCED1 level along with ABA content. Our results provide evidence of the involvement of PtNCED1 in regulating endogenous ABA content in developing seeds and protocorms. The accumulation of endogenous ABA content in orchid seeds may have a critical role in seed dormancy and the protocorm response to water stress after seed germination.
... Shoot division is a slow process and is not conducive for large-scale plant propagation. Asymbiotic seed germination is another method for large-scale propagation of orchids; however, the plants raised by this method are highly heterozygous [7]. Therefore, breeding for traits and creating many individual plants with the same traits are more difficult. ...
Orchids comprise an important group of flowering plants that produce a wide and distinct variety of flowers with characteristic shapes, size, colors, and fragrance. In horticulture industry, orchids are used as cut flowers and as potted plants. Many of the orchids have also seen medicinal use as part of traditional Chinese medicine. To meet the extensive demand for their attractive and showy flowers in the commercial market, tissue culture methods have been developed in many ornamental orchids for their large-scale multiplication. Recently, the bioreactor systems such as airlift, bubble, and temporary immersion bioreactors have been established for large-scale propagation of protocorm-like bodies (PLBs), shoots, and plantlets of various orchids. This chapter describes the bioreactor culture methods for micropropagation of Anoectochilus formosanus and Dendrobium candidum. Methods of estimation of total phenolics, flavonoids, polysaccharides, and antioxidant activities of biomass produced from Dendrobium candidum are also discussed.
... ASYMBIOTIC seed germination has emerged as an important tool for propagating a large number of orchid species and hybrids 1 . The orchid seed can also germinate in vitro prior to reaching maturity. ...
... As regards artificial seed germination, orchids can be classified into three categories (Arditti, 1967;1979;Arditti and Ernst, 1984). Terrestrial, temperate zone orchids are more difficult to germinate asymbiotically (Arditti, 1982) than the tropical ones. By ex situ, in vitro germination, it is very important, that the core must be unripe on the plant when we collect the seeds, because the seeds are only sterile until the cores are closed (Fast, 1980). ...
The germination of the orchids is the most sensitive period of their life. In case of the extremely rare Himantoglossum adriaticum in Hungary, no such information has been published yet. Our aim is to present the results of the in situ and the ex situ germination of H. adriaticum seeds originate in West Hungary. There were 10-10 seed packets sowed 10-20 cm far-, and 3-3 as control 10 m away from the living mother plants on the natural habitats of Keszthelyi-hills and of Sümeg-Tapolca region. The success of germination was significantly higher close to the mother plant than in the control packets either 8 or 11 months after sowing on both places and was a bit better in Keszthely than on Sümeg-Tapolca region. Parallelly with it we sowed the seeds of same origin into six flasks, onto Fast medium in the orchid laboratory of Eötvös Loránd University’s Botanical Garden in Budapest. During this ex situ germination, the first protocorm was appeared 9 months after sowing. The germination was outstanding only in one flask. Our results show, that the germination in the natural habitat was much higher than in vitro on the generally used artificial media.
... Mikoriza menyediakan nutrisi, vitamin, dan hormon pada tanaman muda yang diperlukan sampai tanaman tersebut cukup besar dan mampu memproduksi nutrisinya sendiri (Harrison 1977;Harrison & Arditti 1978). Jika biji telah berkecambah, maka biji tersebut akan menghasilkan massa sel yang tidak mengalami diferensiasi yang disebut protokorm (Arditti 1982). Protokorm ini akan tumbuh setelah beberapa minggu, bulan, bahkan tahunan tergantung spesies, sampai tanaman tersebut cukup besar untuk memproduksi daun dan akar. ...
Konservasi anggrek spesies alam merupakan langkah penting untuk menghindari kepunahan akibat rusaknya habitat alamnya. Tujuan penelitian ialah mengetahui kemampuan tumbuh biji anggrek spesies alam pada media Vacin dan Went (VW) dan menentukan jenis anggrek spesies yang telah berhasil dikonservasi melalui biji. Anggrek spesies alam yang digunakan sebanyak 46 spesies yang berasal dari 18 genus anggrek, yakni Phalaenopsis, Dendrobium, Vanda, Ascocentrum, Paphiopedilum, Rhyncostilis, Neofinetia, Epidendrum, Arachnis, Dimorphosis, Phaius, Spathogottis, Trichoglottis, Arundina, Cymbidium, Renanthera, Armodorum, dan Gramathophylum. Polinasi bunga anggrek dilakukan dengan metode selfing maupun sibling. Buah dipanen saat warna buah berubah menjadi kuning kehijauan dan/atau tekstur buah menjadi lebih lunak. Selanjutnya, sebelum kultur biji, buah disterilkan dan biji disebar pada media VW. Hasil yang diperoleh menunjukkan bahwa buah anggrek hasil polinasi mengalami kemasakan antara 34-280 hari setelah polinasi. Biji hasil selfing dari 41 anggrek spesies (dari 46) dapat berkecambah pada media VW dengan umur berkecambah berkisar antara 10-69 hari setelah sebar. Sebanyak 19 anggrek spesies alam mampu membentuk protokorm di atas 70% dan semua protokorm mampu membentuk planlet. Selanjutnya, 19 spesies dapat diaklimatisasi dengan kisaran waktu antara 272-552 hari setelah terbentuk protokorm, Sebanyak 16 spesies belum dapat diaklimatisasi karena planlet yang masih kecil, sedang 6 spesies tidak tumbuh. Arundina graminifolia merupakan anggrek yang paling cepat membentuk protokorm dan Grammatophylum sp. merupakan anggrek yang paling lama membentuk protokorm. Biji Ascocentrum aurantiacum, Aerides odorata, Phalaenopsis luddemanniana, P. violacea, dan Cymbidium finlaysonianum tidak mampu membentuk protokorm. Dari penelitian ini diketahui bahwa media VW dapat digunakan untuk konservasi anggrek spesies alam melalui perbanyakan dengan menggunakan biji.
... Elucidating the process of embryo development has been examined using two strategies: rescue of the immature embryo by in vitro culture and development of techniques for somatic embryogenesis. Generally, seed germination of terrestrial orchid species from temperate areas is difficult (Arditti et al., 1982;Rasmussen, 1995). High frequencies of seed germination on medium have been achieved in a limited number of orchid species (Dutra et al., 2007(Dutra et al., , 2009Kane, 2005, 2006;Tsavkelova et al., 2007). ...
To find the characteristics of somatic embryogenesis of orchids and elucidate the mechanism, we had previously established an efficient plant regeneration system via somatic embryogenesis in Dendrobium candidum Wall ex Lindl. In this study, a detailed cytological investigation was carried out on the initiation and developmental process of somatic embryogenesis. Based on our observations, the somatic embryogenesis in D. candidum originated from the transition of an embryonic callus cell to the initial somatic embryo cell, and the somatic embryos initiated from those cells. During the transition process, condensation and devacuolation successively occurred in the cytoplasm of the embryonic callus cells, giving rise to the formation of a typical initial somatic embryo cell with dense cytoplasm and a clear nucleus. One of the two pathways in somatic embryogenesis is the single-cell-derived somatic embryo which is generated from an inner initial somatic embryo cell in embryonic callus and develops into a globular somatic embryo in a way similar to zygotic embryogenesis and then keeps developing into a protocorm-like body (PLB). The other is a multiple-cellderived somatic embryo which is generated from peripheral grouped initial somatic cells in embryonic calli and directly forms globular embryo or multicellular somatic proembryo, lacking the typical early stages of embryogenesis. Both pathways were observed in the somatic embryogenesis system, indicating that the culture system in D. candidum can be a useful tool for investigating the mechanisms underlying orchid embryogenesis. © 2017, American Society for Horticultural Science. All rights reserved.
Orchids are extremely popular as horticultural plants with significant ornamental and economic value. These are highly priced in the national and international markets for their enduring and bewitchingly beautiful flowers. Apart from their high ornamental usefulness, orchids are valued for significant medicinal importance, especially as herbal or ethnomedicine since time immemorial. Considering the importance and present demand in both horticultural and medicinal aspects, there is an urgent need to exploit the species in a very scientific way. But, orchids are now facing the danger of depletion, leading to a scarcity of planting material due to natural and manmade calamities. Conventional vegetative propagation is slow and time-consuming; moreover, orchid seeds are exceedingly small and powdery and cannot germinate easily in nature due to the lack of endosperm. Under the above circumstances, biotechnological approaches enhance the in vitro propagation as well as conservation and mass multiplication of important medicinal orchids has raised high hopes by adopting asymbiotic seed germination, vegetative explants materials, artificial seed technology, production of secondary metabolites, etc. This chapter briefly encompasses the state-of-the-art information on tissue culture-mediated biotechnological interventions framed in some medicinal orchids through micropropagation, along with its societal impacts like ethnomedicinal properties, phytochemistry, and biological activity, that being the need of the hour.KeywordsMedicinal orchidMicropropagationSecondary metabolitesEthnomedicinePhytochemistryBiological activity
A protocol for seed germination and seedling culture of Cymbidium aloifolium (L.) Sw. and C. bicolor Lindl. Was developed. Green “matured” capsules containing seeds were surface sterilized and cultured on various media compositions. Murashige and Skoog (MS) medium with or without 10% coconut water (CW) promoted higher rate of initial seed germination. This was followed by modified Knudson’s C and Phytamax media supplemented with or without 10% CW. Further studies on seedling multiplication revealed that MS medium supplemented with 10% CW was more effective in axillary shoot proliferation. Rooted seedlings were successfully acclimatized to the greenhouse environment. Results of this study demonstrate that Cymbidiums can be successfully propagated through asymbiotic germination method for possible use in re-introduction in situ and conservation projects.
Background
Cypripedium subtropicum is a unique, endangered lady’s slipper orchid with evergreen leaves on non-dormant shoots that is native to southwestern China. This study documents the major developmental events in C. subtropicum seed development from fertilization to seed maturity, determines the optimum period for seed collection, and examines the cytokinin requirements for asymbiotic germination and protocorm survival.
Results
Structural studies revealed that embryo development proceeded after successful fertilization at 60 days after pollination (DAP). At 105 DAP, a globular embryo with the shrinking inner seed coat was observed, and seeds collected at this time point exhibited optimal germination. After 120 DAP, most seeds had a mature embryo within the capsule, and within the cells of the embryo proper, numerous proteins/lipid bodies were present as the main storage products. In addition, the inner seed coat had compressed into a thin layer that tightly enclosed the embryo, while the outer seed coat had progressively elongated, resulting in a hair-like appearance of the mature seed. Histochemical staining using Nile red and toluidine blue O (TBO) indicated that the lignified inner and outer seed coats may lead to coat-imposed dormancy. Seeds collected at this stage germinated poorly. Analyses of cytokinin preferences and optimal concentrations for germination and protocorm survival showed that both 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-benzylaminopurine (BA) enhanced germination compared with the control, although higher concentrations of BA (4 and 8 μM) suppressed germination. The protocorm survival rate improved with increasing 2iP concentration.
Conclusions
This study provides a reproducible procedure for culturing immature seeds of C. subtropicum based on a defined time schedule of seed development. In addition, the cytokinin 2iP was shown to improve germination and protocorm survival. This study provides a scientific basis for seedling establishment through asymbiotic seed culture for further reintroduction efforts.
Plants belonging to the Dendrobium genus occupy a dominant position among the orchids because of their high ornamental and therapeutic values. They are widely popular in the international floriculture trade as they bear stunning flowers with diverse coloration, varied forms, and patterns. The ethnomedicinal uses of these orchids are also prominently found due to the possession of immense medicinal properties. Excessive exploitation through rampant unregulated collection and widespread habitat destruction have dwindled the Dendrobium natural populations at an alarming rate. The fast and reliable micropropagation techniques offer an alternative to the slow conventional methods of Dendrobium propagation. The rapid in vitro regeneration of genetically stable Dendrobiums is essential for effective germplasm conservation and large-scale orchid commercialization. Several genetic stable Dendrobiums have been successfully propagated by ascertaining the clonal fidelity of the regenerants using different molecular markers. This chapter focuses on the uses of Dendrobiums as important ethnomedicine, their in vitro propagation, and clonal assessment for producing genetically stable orchids using DNA markers.
Cymbidiums enjoy a prime position in the floriculture industry for heavy substance of the flowers and high flower longevity. They are cultivated for cut flowers as well as pot plants for house decorations. Besides, they are used in medicine, cosmetics, and food. The successful cymbidium cultivation requires effective management of several factors controlling production and productivity. Various aspects of cymbidium orchid cultivation including genetics and breeding and plant health management have been discussed in the chapter.
Kalimantan whose existence is increasingly extinct so it needs to be preserved by in vitro propagation. This study aims to determine the effect of a combination of the growth regulators, Benzyl Amino Purin (BAP), Kinetin, Naphthalene Acetic Acid (NAA) and banana extract on the growth of sugar cane orchids in vitro. The basic media used were Half Murashige and Skoog (1/2 MS), on the base media were added 30 g.L-1 sugar, 8 g.L-1 seaweed commercial powder, plus BAP 3 ppm, Kinetin 3 ppm, NAA 0.5 ppm, and ambon banana extract 100 g.L-1, the acidity (pH) of the media is adjusted to around 5.8. The research design used was a Completely Randomized Design (CRD), each treatment was repeated six times. Data were analyzed using variance analysis and continued with DMRT (Duncan Multiple Range Test) tests with a level of 5%. The results showed that the half media Murashige and Skoog was good for the propagation of sugarcane orchids. The addition of BAP growth regulators, Kinetin, NAA, and banana extracts showed a good response to growth culture. The addition of BAP 3 ppm + NAA 0.5 ppm showed the best results for the height increased, leaf number increased, leaf length increased, leaf width increased, and the number of shots increased. The addition of Ambon banana extract to media containing growth regulators tended to decrease the growth response, seen in the parameters of plant height increased, leaf number increased, leaf length increased, leaf width increased, the number of shoots increased, the number of roots increased and in root length increased.
Dactylorihza umbrosa is a species of native terrestrial orchid in Iran that is important as an ornamental and medicinal plant. Salap is extracted from their underground organ, which is very valuable in the pharmaceutical and food industries. This plant is endangered due to the improper harvesting of its underground organs. Mass propagation with new methods can be a good solution to protect this valuable plant from extinction. In this study, different kind of carbohydrate-containing sucrose, fructose, and glucose (0, 20, 30 g/l) and organic compounds containing peptone and casein hydrate (0, 2 g/l) in FAST as a basal medium were examined for their effectiveness in non-symbiotic germination rate and development of an Iranian native orchid species Dactylorihza umbrosa in darkness condition. Results showed that the treatments of carbohydrates, organic compounds, and the interaction between them had a significant effect on germination percentage. Casein has a good effect on embryogenesis in different carbohydrate source treatments. The highest germination with 98.07 % was observed in FAST-medium containing 20 gr/l glucose and 2 g/l casein hydrate. Mature protocorms were transferred to the culture medium containing 0 and 2 g / l activated charcoal under light conditions after four months. The positive effect of treatment with activated charcoal 2 g / l in the rate of growth of shoots and roots of seedlings was observed.
Growth and morphogenesis of orchid in vitro culture is influenced by supplementation with certain undefined additives. Various organic compounds, i.e., coconut water, banana and potato homogenate, carrot and tomato juice, casein hydrolysate, peptone, yeast extract, chitosan and phloem sap, individually or in combination, can promote seed germination, seedling development, protocorm-like bodies (PLBs) formation, plantlet growth, and multiple shoot regeneration of various orchids. These organic supplements are a source of minerals, vitamins, amino acids, organic acids, carbohydrates, peptides, growth regulators or their derivatives, and antioxidants. The amount of these substances required for successful orchid in vitro culture varies with the species and genotype.
Orchids are known for their aesthetic qualities, and they are often used as decorative items in homes, offices, and public places. While most people admire them for their good looks, others have found practical uses for them. Since a long time ago, people from various parts of the world have used orchids for medicinal purposes. However, the use of orchids in medicine has declined over the years because not enough research has been done to determine their effectiveness and adverse effects.
Gastrodia elata is a well-known medicinal and heterotrophic orchid. Its germination, limited by the impermeability of seed coat lignin and inhibition by abscisic acid (ABA), is triggered by symbiosis with fungi such as Mycena spp. However, the molecular mechanisms of lignin degradation by Mycena and ABA biosynthesis and signaling in G. elata remain unclear. In order to gain insights into these two processes, this study analyzed the transcriptomes of these organisms during their dynamic symbiosis. Among the 25 lignin-modifying enzyme genes in Mycena, two ligninolytic class II peroxidases and two laccases were significantly upregulated, most likely enabling Mycena hyphae to break through the lignin seed coats of G. elata. Genes related to reduced virulence and loss of pathogenicity in Mycena accounted for more than half of annotated genes, presumably contributing to symbiosis. After coculture, upregulated genes outnumbered downregulated genes in G. elata seeds, suggesting slightly increased biological activity, while Mycena hyphae had fewer upregulated than downregulated genes, indicating decreased biological activity. ABA biosynthesis in G. elata was reduced by the downregulated expression of 9-cis-epoxycarotenoid dioxygenase (NCED-2), and ABA signaling was blocked by the downregulated expression of a receptor protein (PYL12-like). This is the first report to describe the role of NCED-2 and PYL12-like in breaking G. elata seed dormancy by reducing the synthesis and blocking the signaling of the germination inhibitor ABA. This study provides a theoretical basis for screening germination fungi to identify effective symbionts and for reducing ABA inhibition of G. elata seed germination.
For many reasons tobacco is extensively used as a model plant in transformation research and used for this study as well. Tobacco plants are cultivated in several countries on large scale. Tobacco has numerous essential traditional and modern uses in clinical field. The principle aim of this present study was to optimize the conditions for seed germination, best shooting regeneration media, optimization of antibiotics spectinomycin and kanamycin for wild type plants and in addition, to develop a protocol for efficient genetic transformation of tobacco plant cv. Petit Havana with Agrobacterium tumefaciens. Seeds of tobacco were germinated on different media without any plant growth regulators. 100% seeds germination efficiency was observed on ½ MS and best sterilization time of tobacco with ethanol was for 1 minute which also showed 100% germination efficiency. RMOP (NAA and BAP) media showed the highest shoots regeneration efficiency for both explants nodes and leaves which is nodes showed 75% and leaves showed 55% regeneration efficiency among the different media which is used for regeneration. 150 mg/L concentration of kanamycin and 500 mg/L concentration of spectinomycin for both explants (nodes and leaves) was optimized for the selection of transgenic tissues. For transformation infection time for 5 minutes and co-cultivation time period of two days showed maximum transformation efficiency of 83.33% as compared to other methods. Histochemical GUS (β-glucuronidase) assay was used to check the GUS gene expression in the nodal explants. GUS gene primers were used which amplified a 708bp fragment which also confirmed the transformation of GUS gene by Agrobacterium tumifaciens strain C58C1 containing vector p35SGUSINT. Taken together, the present study develops a tissue culture protocol and basis for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Petit Havana using nodal explants.
Keywords: Tobacco, Spectinomycin, Kanamycin, Agrobacterium tumefaciens, GUS, Infection time, Co-cultivation time.
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