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

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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Asymbiotic in vitro germination and seed quality assessment of Australian terrestrial orchids

Australian Journal of Botany ◽

10.1071/bt12133 ◽

2012 ◽

Vol 60 (7) ◽

pp. 592 ◽

Cited By ~ 11

Author(s):

Nicole Dowling ◽

Manfred Jusaitis

Keyword(s):

Seed Quality ◽

Culture Media ◽

Vital Staining ◽

Terrestrial Orchid ◽

Triphenyltetrazolium Chloride ◽

Asymbiotic Seed Germination ◽

Orchid Species ◽

Terrestrial Orchids ◽

Asymbiotic Germination

Determining the seed quality and germination requirements for threatened orchid species in storage is vital for future conservation efforts. Seeds of many Australian terrestrial orchid species are held in conservation collections around the country, but few have been germinated in vitro, fuelling concerns over their long-term viability. This study tested three methods of assessing orchid seed quality; asymbiotic germination was compared with vital staining using triphenyltetrazolium chloride or fluorescein diacetate. Six culture media were examined for efficacy in promoting asymbiotic seed germination of four Australian terrestrial orchid species (Pterostylis nutans, Microtis arenaria, Thelymitra pauciflora and Prasophyllum pruinosum). Germination occurred on all media but germination rates were consistently highest on BM1 and development was most advanced on BM1, P723 and Malmgren media. Subsequent trials tested the efficacy of BM1 for asymbiotic germination of additional genera (Caladenia, Calochilus and Diuris), several congeneric species, and two species collected from several different provenances within each of their ranges. The results indicate that asymbiotic germination on BM1 medium is an effective technique for testing the performance of Australian terrestrial orchid seeds. The efficacy of vital stains to determine seed viability, however, remains uncertain, as significant disagreement between degree of staining and germinability was observed for some 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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Germination and Protocorm Formation of Ophrys Sphegodes Mill. – In Vitro Protocol for a Rare Orchid Species

Contemporary Agriculture ◽

10.1515/contagri-2018-0028 ◽

2018 ◽

Vol 67 (3-4) ◽

pp. 196-201

Author(s):

Jovana Dulić ◽

Mirjana Ljubojević ◽

Ines Prlainović ◽

Goran Barać ◽

Tijana Narandžić ◽

...

Keyword(s):

Seed Germination ◽

Germination Rate ◽

Casein Hydrolysate ◽

Habitat Destruction ◽

Ex Situ ◽

Organic Additives ◽

Orchid Species ◽

Short Period ◽

Protocorm Formation

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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Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010005161x ◽

1974 ◽

Vol 32 ◽

pp. 320-321

Author(s):

J. P. Revel

Keyword(s):

Developmental Stages ◽

Three Dimensional ◽

Cell Movement ◽

Cellular Interactions ◽

Serial Sections ◽

Cell Sheets ◽

Freeze Etching ◽

Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

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Pertumbuhan Dan Perkembangan Anggrek Dendrobium anosmum Pada Media Kultur In Vitro Dengan Beberapa Konsentrasi Air Kelapa

Agrologia ◽

10.30598/a.v1i1.293 ◽

2018 ◽

Vol 1 (1) ◽

Author(s):

S. Tuhuteru ◽

Meity L Hehanussa ◽

Simon H.T Raharjo

Keyword(s):

Culture Medium ◽

Culture Media ◽

Water Concentration ◽

Medium Composition ◽

Coconut Water ◽

Orchid Species ◽

Randomized Design ◽

Wet Weight ◽

Completely Randomized Design

Dendrobium anosmum is one of natural orchids in Indonesia. Optimization of medium composition for orchid propagation through in vitro culture is necessary to enhance propagule multiplication capabilities and quality. This study was aimed to study the influence of concentration of coconut water in culture medium on in vitro growth and development of D. anosmum orchid species and to determine the optimal coconut water concentration in culture media. The experiment were arranged in a Completely Randomized Design with four treatments and eight replications. The treatments consisted of the addition of coconut water with concentrations: 0 ml•l -1 (control), 50 ml•l-1, 100 ml•l-1 and 150 ml•l-1. The results showed that addition of coconut water in culture medium gave different effect on shoot growth and multiplication of D. anosmum orchids. Coconut water concentration of 100 ml•l-1 was the best concentration for growth and multiplication of D. anosmum orchids, based on both shoots and roots growth, plantlet height and wet weight.

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Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽

10.3390/plants10040776 ◽

2021 ◽

Vol 10 (4) ◽

pp. 776

Author(s):

Shipra Kumari ◽

Bashistha Kumar Kanth ◽

Ju young Ahn ◽

Jong Hwa Kim ◽

Geung-Joo Lee

Keyword(s):

Abiotic Stress ◽

Biotic Stress ◽

Developmental Stages ◽

Expression Patterns ◽

Lilium Longiflorum ◽

Biotic And Abiotic Stress ◽

Biotic Stresses ◽

Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

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Zika Virus Infection Leads to Demyelination and Axonal Injury in Mature CNS Cultures

Viruses ◽

10.3390/v13010091 ◽

2021 ◽

Vol 13 (1) ◽

pp. 91

Author(s):

Verena Schultz ◽

Stephanie L. Cumberworth ◽

Quan Gu ◽

Natasha Johnson ◽

Claire L. Donald ◽

...

Keyword(s):

Nervous System ◽

Neural Development ◽

Zika Virus ◽

Developmental Stages ◽

Cell Types ◽

Neural Cells ◽

Zikv Infection ◽

Axonal Pathology ◽

Time Frames

Understanding how Zika virus (Flaviviridae; ZIKV) affects neural cells is paramount in comprehending pathologies associated with infection. Whilst the effects of ZIKV in neural development are well documented, impact on the adult nervous system remains obscure. Here, we investigated the effects of ZIKV infection in established mature myelinated central nervous system (CNS) cultures. Infection incurred damage to myelinated fibers, with ZIKV-positive cells appearing when myelin damage was first detected as well as axonal pathology, suggesting the latter was a consequence of oligodendroglia infection. Transcriptome analysis revealed host factors that were upregulated during ZIKV infection. One such factor, CCL5, was validated in vitro as inhibiting myelination. Transferred UV-inactivated media from infected cultures did not damage myelin and axons, suggesting that viral replication is necessary to induce the observed effects. These data show that ZIKV infection affects CNS cells even after myelination—which is critical for saltatory conduction and neuronal function—has taken place. Understanding the targets of this virus across developmental stages including the mature CNS, and the subsequent effects of infection of cell types, is necessary to understand effective time frames for therapeutic intervention.

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Design, Synthesis and Antiparasitic Evaluation of Click Phospholipids

Molecules ◽

10.3390/molecules26144204 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4204

Author(s):

George E. Magoulas ◽

Pantelis Afroudakis ◽

Kalliopi Georgikopoulou ◽

Marina Roussaki ◽

Chiara Borsari ◽

...

Keyword(s):

Leishmania Donovani ◽

Developmental Stages ◽

Neglected Tropical Diseases ◽

Heterocyclic Ring ◽

Head Group ◽

Ether Phospholipid ◽

Design Synthesis ◽

Antiparasitic Activity ◽

Intracellular Amastigotes

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure–activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 μM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.

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Development of an embryo germination protocol for shy-seeded grape (Vitis vinifera L.)

Plant Genetic Resources ◽

10.1017/s1479262121000307 ◽

2021 ◽

pp. 1-9

Author(s):

Ashwini P. Benke ◽

Ram Krishna ◽

Roshni R. Samarth ◽

Shweta S. Dhumal ◽

Waquar A. Ansari ◽

...

Keyword(s):

Developmental Stages ◽

Germination Rate ◽

Germination Percentage ◽

Vitis Vinifera L ◽

Growth Media ◽

Embryo Germination ◽

Indole Butyric Acid ◽

Grape Berries ◽

Benzyl Amino Purine

Abstract Acquisition and germination of seeds are the most desired targets for the improvement of vegetatively propagated crops. In the present study, we developed a potential embryo germination protocol for the Red Globe grape cultivar having a low seed germination rate. Three grape berries at different developmental stages, viz. 50, 60 and 70 days after flowering (DAF), were selected for in-vitro embryo germination. Three growth media, namely Emershad and Ramming (ER), Nitsch and Nitsch (NN) and Murashige and Skoog (MS), and plant growth regulators (benzyl amino purine (BA), 0.5, 0.7 and 0.9 mg/l; indole butyric acid (IBA), 1.0, 1.5 and 2.0 mg/l; and gibberellic acid (GA), 0.1, 0.3 and 0.9 mg/l) were screened individually in different combinations with three amino acids, namely cysteine, glutamine and proline (2.0 μmol/l each). The maximum embryos germination percentage recorded at 70 DAF was 63.33, 47.78 and 45.56% in ER, NN and MS media, respectively, supplemented with 0.9 mg/l BA, 2.0 mg/l IBA, 0.9 mg/l GA and 2.0 μmol glutamine. Glutamine was found to have the most significant impact, and it improved the rescued embryos germination. The present study provides a potential recipe for a medium that can facilitate efficient germination of grape embryos.

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Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽

10.1017/s0967199419000832 ◽

2020 ◽

Vol 28 (2) ◽

pp. 154-159

Author(s):

Juliana I. Candelaria ◽

Anna C. Denicol

Keyword(s):

Granulosa Cells ◽

Developmental Stages ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Cell Number ◽

Culture Conditions ◽

Preantral Follicles ◽

Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

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