Germination and Protocorm Formation of Ophrys Sphegodes Mill. – In Vitro Protocol for a Rare Orchid Species

Summary Ophrys sphegodes Mill. is a wild orchid species which is threatened and protected due to its pollination biology, small seed and habitat destruction. The aim of this study was to establish asymbiotic germination protocol for the purpose of ex situ conservation. Two basal media Knudson C (KC) and Malmgren (MM), supplemented with organic additives (peptone (PE), L-glutamin (A)e, folic acid, casein hydrolysate (CA)) added separately and control media KC--C and MM--C were used in the present research. All the nutrition media contained 2% sucrose, 7% agar and 1% activated carbon, while their pH was adjusted to 5.8 ± 0.02 before autoclaving at 121 ° C for 20 minutes. The seeds were examined under two illumination conditions, 0/24 light/dark (L/D) and 16/8 L/D. The presented results indicate a huge influence of illumination and nutrition media on the seed germination and protocorm formation. The seed germination was overall significantly more successful in dark conditions (0/24 L/D) than with lighting (16/8 L/D). Protocorm, rhizoids and shoot formation were achieved only on the seeds cultured on MM medium, while the KC medium caused only swelling of the embryo. Organic additives had positive influence on the germination rate. According to the obtained results, the best germination rate and seedling development were achieved on MM-PE media, cultured in dark. The presented procedure accelerates the germination period and can provide a large number of plants in a relatively short period of time so it can be used for conservation programs and mass production protocol.

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A Review on Asymbiotic Seed Germination in Orchids through Plant Tissue Culture

10.54280/21/05 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Purnima Paramanik ◽

◽

Dipak Kar ◽

Subrata Raha ◽

◽

...

Keyword(s):

Seed Germination ◽

Mineral Nutrients ◽

Symbiotic Association ◽

Organic Additives ◽

Asymbiotic Seed Germination ◽

Orchid Species ◽

Mycorrhizal Association ◽

Commercially Important

Orchid seeds are very small, dust like in appearance, fusiform in shape, lacking endosperm and have undifferentiated embryo covered by transparent seed coat. Mycorrhizal association is required for seed germination of naturally growing orchids. In this symbiotic association, orchid species are dependent on mycorrhizal association for supply of mineral nutrients. In in-vitro condition, such demand of minerals may be compensated by external supply of sugar and mineral nutrients that are required for seed germination of orchid. Several orchids are responded by this asymbiotic seed culture and have commercial importance. Therefore, formulation of efficient in-vitro protocol is important for commercially important orchid species as well as endangered orchid species for conservation. This review paper is focused on various aspects of asymbiotic seed germination of orchids and the role of organic additives in successful seed germination.

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ASYMBIOTIC GERMINATION OF CYPRIPEDIUM CALCEOLUS VAR. PARVIFLORUM (ORCHIDACEAE) IN VITRO.

HortScience ◽

10.21273/hortsci.27.6.618d ◽

1992 ◽

Vol 27 (6) ◽

pp. 618d-618

Author(s):

Chin-Chang Chu ◽

Kenneth W. Mudge

Keyword(s):

Seed Germination ◽

Suspension Culture ◽

Agar Medium ◽

Germination Rate ◽

Coconut Water ◽

Mature Seed ◽

Orchid Species ◽

Cypripedium Calceolus ◽

Chilling Treatment

Seeds from mature seed pods of Cypripedium calceolus var. parviflorum were germinated on 1/4 MSMO (Sigma) + 100ml/l coconut water + 1% sucrose +/- 8g/l agar (pH 6.0), and with or without prechilling at 5C for 8 weeks. Protocorm with apex (stage 3) was use as an index of germination. Seeds sown on agar medium withou chilling treatment resulted in a 40% germination rate in 120 days but the germination was very uneven. Seeds germinated on agar medium with prechilling developed more synchronously with 92% germination in 60 days (ie. about 120 days after sowing). Suspension culture of seeds without prechilling resulted in 85% germination after 90 days. The synchronization of seed germination in suspension culture was intermediate between that on agar with and without prechilling. Protocorms germinated in suspension culture appeared morphologically identical to those germinated on agar medium. All stage 3 protocorms developed further on the same agar medium in darkness. Both agar and suspension culture in media containing coconut water provided reliable seed germination methods for this orchid species.

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In vitro seed germination in Cymbidium elegans Lindl. and Dendrobium densiflorum Lindl. ex Wall. (Orchidaceae)

Botanica Orientalis Journal of Plant Science ◽

10.3126/botor.v6i0.2917 ◽

2010 ◽

Vol 6 ◽

pp. 100-102 ◽

Cited By ~ 5

Author(s):

Shreeti Pradha ◽

Bijaya Pant

Keyword(s):

Seed Germination ◽

Basal Medium ◽

Naphthalene Acetic Acid ◽

Genetic Constitution ◽

Ms Medium ◽

Orchid Species ◽

C Elegans ◽

Protocorm Formation ◽

Dendrobium Densiflorum

A comparative study of in vitro seed germination of two endangered orchid species, viz. Cymbidium elegans Lindl. and Dendrobium densiflorum Lindl. ex Wall., was carried out on Murashige and Skoog's (MS) medium, supplemented with different concentrations and combination of 6-benzylaminopurine (BAP) and á-Naphthalene acetic acid (NAA). The hormone-free MS medium and MS medium supplemented with various growth hormones were found effective for in vitro seed germination of both species. However, the seeds of these two species showed variation in their germination behavior. Hormone-free MS basal medium was found most effective for seed germination of D. densiflorum; whereas, basal medium supplemented with BAP (1mg/l) was effective for C. elegans. The seeds of D. densiflorum showed quick response in earlier germination, protocorm formation and further development into seedlings in comparison to C. elegans. In C. elegans, germination of immature seeds started after nine weeks of inoculation; whereas in D. densiflorum, the initiation of germination started after five weeks of culture. The variations in seed germination, protocorm formation and seedling differentiation in the two orchid species might be due to the differences in their genetic constitution and the presence of different endogenous growth stimulating substances present in their seeds. The present study has provided useful information for in vitro clonal mass multiplication of these commercially important orchid species. Key-words: growth hormone; in vitro study; orchid.DOI: 10.3126/botor.v6i0.2917 Botanica Orientalis - Journal of Plant Science (2009) 6: 100-102

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FEATURES SEED REPRODUCTION OF HEDYSARUM GRANDIFLORUM PALL. EX SITU AND IN VITRO

Вестник Пермского университета. Серия «Биология»=Bulletin of Perm University. Biology ◽

10.17072/1994-9952-2020-4-286-293 ◽

2020 ◽

pp. 286-293

Author(s):

N. A. Suprun ◽

◽

E. V. Malaeva ◽

S. A. Shumikhin ◽

◽

...

Keyword(s):

Seed Germination ◽

Germination Rate ◽

Natural Populations ◽

Seed Vigor ◽

Ex Situ ◽

Optimal Temperature ◽

High Productivity ◽

Seed Reproduction ◽

Optimal Mode

Hedysarum grandiflorum samples were collected from 7 natural populations of the Volgograd, Samara and Lugansk People's Republic and the features of seed reproduction were studied under ex situ and in vitro conditions. The optimal temperature for seed germination and the dependence of germination and seed vigor on the storage were revealed. We observed the maximum germination rate in the scarified seeds of H. grandiflorum of 2018 collection and it was equal to 70%.It was found that the seed germina-tion and seed vigor are low and quickly decrease with years of storage, while their scarification allows to increase these indicators. It is noted that the relatively low germination of the studied seeds is leveled by the rather high productivity of species in general. The optimal mode of sterilization of H. grandiflorum seeds was experimentally determined - 10% Lysofornin® 3000 solution for 5 minutes. The percentage of sterile seedlings was 60%.

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The Stimulating Effects of Imazalil and Carbendazim Fungicides on in Vitro Propagation and ex Situ conservation of the Medicinal Balkan Range-Restricted Sideritis Raeseri Boiss & Heldr. Subsp. Raeseri

JOURNAL OF ADVANCES IN AGRICULTURE ◽

10.24297/jaa.v10i0.8282 ◽

2019 ◽

Vol 10 ◽

pp. 1752-1763 ◽

Cited By ~ 1

Author(s):

Virginia Sarropoulou ◽

Eleni Maloupa

Keyword(s):

Large Scale ◽

Ex Situ Conservation ◽

Germination Rate ◽

Shoot Multiplication ◽

Shoot Length ◽

Ex Situ ◽

Root Number ◽

Short Period ◽

Shoot Number

Sideritis raeseri Boiss & Heldr. subsp. raeseri, known in Greece as Mountain tea of Parnassus or Velouchi is a range restricted medicinal plant of the Balkan peninsula. Conventional propagation methods did not allow the mass production of plant material in a short period of time due to both low seed germination rate and rooting of cuttings. Therefore, the aim of this study was to establish a reliable, reproducible and efficient regeneration protocol for mass and large-scale micropropagation, germplasm and ex situ conservation of S. raeseri Boiss & Heldr. using Imazalil and Carbendazim fungicides. After 9 weeks, 2.5-10 mg/l Imazalil stimulated root length by 1 cm but diminished root number and rooting percentage. Optimum shoot number (5.58), shoot length (24.91 mm), shoot multiplication (100%), root number (20.63) and rooting (66.67%) were recorded with 0.5 mg/l kinetin (KN) + 0.05 mg/l ?-napthaleneacetic acid (NAA) (Imazalil-free). After 4 ½ weeks, 1 mg/l Carbendazim + 0.5 mg/l 6-benzyladenine (BA) yielded 5.77 shoots/explant, 16.1 mm shoot length and 100% shoot multiplication. However, Carbendazim did not result in rooting. The ex vitro survival percentage of rooted shoot-tip explants derived from Imazalil experiment was 95%. Carbendazim proved useful in shoot proliferarion and Imazalil in root elongation of S. raeseri Boiss & Heldr. subsp. raeseri micropropagation system.

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Organic Additives Improves the in Vitro Growth of Native Orchid Vanda helvola Blume

Notulae Scientia Biologicae ◽

10.15835/nsb729546 ◽

2015 ◽

Vol 7 (2) ◽

pp. 192-197

Author(s):

Devina DAVID ◽

Roslina JAWAN ◽

Hartinie MARBAWI ◽

Jualang Azlan GANSAU

Keyword(s):

Seed Germination ◽

Rapid Development ◽

Basal Medium ◽

Tomato Juice ◽

Organic Additives ◽

Orchid Species ◽

Orchid Conservation ◽

Hand Pollination ◽

Basal Media

In vitro seed germination has been proven to be the most efficient technique to propagate orchid. The application of this aseptic technique has contributed to conservation of many endangered orchid species. In this study, undehisced capsules of Vanda helvola Blume were collected from Orchid Conservation Centre in Lagud Sebrang Agriculture Park, after 120 days from hand pollination and aseptically cultured on three types of basal media such as Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW). After 90 days of culture, 66.40 ± 4.14% of seeds successfully germinated on KC medium. The effect of organic additives such as tomato juice, coconut water, peptone and yeast extract at different level of concentrations in KC basal medium were also tested on seed germination and seedling development of this native orchid. After 90 days of culture, over 90% of seeds were tremendously germinated on KC medium supplemented with 10% or 15% (v/v) of tomato juice. The incorporation of peptone at 0.1% (w/v) in KC basal media promoted rapid development of protocorm to seedling. Seedlings on this treatment produced an average of three leaves and two roots after 90 days of culture and were successfully acclimatized.

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Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

PLoS ONE ◽

10.1371/journal.pone.0243297 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243297

Author(s):

Franciele Marx Koene ◽

Érika Amano ◽

Eric de Camargo Smidt ◽

Luciana Lopes Fortes Ribas

Keyword(s):

Developmental Stages ◽

Morphometric Study ◽

Morphometric Characters ◽

Orchid Species ◽

Morphological Studies ◽

Germination Process ◽

Length Width ◽

Asymbiotic Germination ◽

Protocorm Formation

The morphological and morphometric characters of seeds belonging to 11 species of the subtribe Pleurothallidinae using light and scanning electron microscopy were studied to understand the in vitro germination process. Qualitative data (color, shape, ornamentation) and quantitative ones were also evaluated in seeds and embryos (length, width, volume and air space percentage between the integument and the embryo). The viability of the seeds was evaluated by in vitro germination in woody plant medium (WPM), and by analysis of the developmental stages of protocorms until seedling formation (two to 24 weeks). Morphometric data showed variations within the genus Acianthera and between species of different genera. The best germination and protocorm formation responses occurred with Acianthera prolifera (92%) and Acianthera ochreata (86%), with the formation of seedlings after 12 and 16 weeks of sowing, respectively. The seeds and embryos of A. prolifera and A. ochreata were larger (length, width, and volume) with a structural polarity that may have facilitated their germination comparing to others studied species. Other characteristics of A. prolifera seeds that may have contributed to these results include the presence of a thin testa without ornamentation and a suspensor. The protocorms of Anathalis obovata, Dryadella liliputiana, and Octomeria gracillis developed slowly in the WPM, not reaching the seedling stage in 24 weeks of cultivation. This morphological and morphometric study contributes to the understanding of asymbiotic germination of some micro-orchid species.

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In vitro seed germination and seedling development of Phaius tancarvilleae (L’Her.) Blume.

Scientific World ◽

10.3126/sw.v9i9.5518 ◽

1970 ◽

Vol 9 (9) ◽

pp. 50-52 ◽

Cited By ~ 3

Author(s):

Bijaya Pant ◽

Sumitra Shrestha ◽

Shreeti Pradhan

Keyword(s):

Acetic Acid ◽

Seed Germination ◽

Seedling Development ◽

Naphthalene Acetic Acid ◽

Ideal Condition ◽

Ms Medium ◽

Mass Propagation ◽

Asymbiotic Seed Germination ◽

Protocorm Formation

In vitro seed germination and seedling development of Phaius tancarvilleae (L’Her.) Blume. was carried out on 0.8%(w/v) agar solidified MS Medium (Murashige and Skoog, 1962) without hormones or supplemented with different concentration and combination of Naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP). MS medium supplemented with 0.5 mg/l of BAP was the most ideal condition for early seed germination, protocorm formation and development of seedlings. Germination started after 7 weeks of culture and complete seedlings were obtained after 24 weeks of culture. This protocol might be helpful for mass propagation of orchids by asymbiotic seed germination. Keywords: Orchid; Invitro; Protocorm; Media DOI: http://dx.doi.org/10.3126/sw.v9i9.5518 SW 2011; 9(9): 50-52

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Establishment of an EfficientIn VitroRegeneration Protocol for Rapid and Mass Propagation ofDendrobium chrysotoxumLindl. Using Seed Culture

The Scientific World JOURNAL ◽

10.1155/2014/740150 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 9

Author(s):

Potshangbam Nongdam ◽

Leimapokpam Tikendra

Keyword(s):

Acetic Acid ◽

Seed Germination ◽

Germination Percentage ◽

Naphthalene Acetic Acid ◽

Mass Propagation ◽

Asymbiotic Seed Germination ◽

Orchid Species ◽

Seed Culture ◽

Leaf Formation

An efficientin vitroregeneration protocol from seed culture has been established successfully forDendrobium chrysotoxum, an epiphytic orchid having tremendous ornamental and medicinal values. Seed germination response was encouraging in Mitra (M) medium enriched with different combinations of auxins and cytokinins. Medium supplemented with 0.4% activated charcoal (AC), 2 mg/L 6-benzyl amino purine (BAP), and 2 mg/L indole-3-acetic acid (IAA) produced best seed germination percentage in 2 weeks of culture. Incorporation of higher concentration of kinetin (KN) or BAP in combination with low auxin in medium induced pronounced shooting and leaf formation. Reduction in leaf development was evident when cytokinins exist singly in medium indicating synergistic effect of auxin and cytokinin in leaf induction. Presence of elevated level of indole-3-butyric acid (IBA) or 1-naphthalene acetic acid (NAA) with low cytokinin content in medium generated morein vitrorooting, though IBA was found to be more effective in rooting induction as compared to NAA. Thein vitroprotocol for asymbiotic seed germination developed from the present investigation can be used for rapid mass propagation of this highly importantDendrobiumorchid species.

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The influence of light spectra, UV-A, and growth regulators on the in vitro seed germination of Senecio cineraria DC.

Revista CERES ◽

10.1590/s0034-737x2010000500002 ◽

2010 ◽

Vol 57 (5) ◽

pp. 576-580 ◽

Cited By ~ 1

Author(s):

Cristiane Pimentel Victório ◽

Nina Cláudia Barbosa da Silva ◽

Maria Apparecida Esquibel ◽

Alice Sato

Keyword(s):

Seed Germination ◽

Growth Regulators ◽

White Light ◽

Germination Rate ◽

Germination Percentage ◽

Ms Medium ◽

In Vitro Germination ◽

Light Spectra ◽

Indole 3 Acetic Acid

This study was carried out to investigate the effects of light spectra, additional UV-A, and different growth regulators on the in vitro germination of Senecio cineraria DC. Seeds were surface-sterilized and inoculated in MS medium to evaluate the following light spectra: white, white plus UV-A, blue, green, red or darkness. The maximum germinability was obtained using MS0 medium under white light (30%) and MS + 0.3 mg L-1 GA3 in the absence of light (30.5%). S. cineraria seeds were indifferent to light. Blue and green lights inhibited germination. Different concentrations of gibberellic acid (GA3) (0.1; 0.4; 0.6; 0.8; 1.0 and 2.0 mg L-1) and indole-3-acetic acid IAA (0.1; 0.3 and 1.0 mg L-1) were evaluated under white light and darkness. No concentration of GA3 enhanced seed germination percentage under white light. However, when the seeds were maintained in darkness, GA3 improved germination responses in all tested concentrations, except at 1.0 mg L-1. Under white light, these concentrations also increased the germination time and reduced germination rate. Germination rate, under light or darkness, was lower using IAA compared with GA3.

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