scholarly journals Regeneration of Viburnum dentatum L. from Alginate-Encapsulated Shoot Explants after Short-Term Cold Storage and Assessment of Genetic Stability Using ISSR Analysis

Agronomy ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1660
Author(s):  
Stefanos Hatzilazarou ◽  
Stefanos Kostas ◽  
Maria Joachim ◽  
Athanasios Economou
Keyword(s):  
Calcium Chloride ◽  
Cold Storage ◽  
Genetic Stability ◽  
Nutrient Medium ◽  
Mother Plant ◽  
Artificial Seeds ◽  
Inter Simple Sequence Repeats ◽  
Regeneration Response ◽  
Regeneration Procedure

The present study demonstrates an efficient protocol for alginate encapsulation, interim cold storing of artificial seeds and conversion to genetically stable plants of Viburnum dentatum L. “Lucidum Aiton”. Explants of shoot tips and first-node segments, excised from in vitro-derived viburnum microshoots, were encapsulated in 2.5% sodium alginate mixed with liquid MS nutrient medium and hardened in 50 mM of calcium chloride producing solid, soft and uniform beads. These artificial seeds achieved 28.9% germination under light, forming 4.3 microshoots per bead. However, with 100 mM of calcium chloride for hardening, the beads were firm and of a uniform globular shape and suitable for handling and exhibited a germination response of 48.9%. Encapsulated shoot tip explants of viburnum, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in regeneration response (73.3, 62.2, 51.1%, respectively), while non-encapsulated explants, stored under same conditions, did not survive after the fourth week of cold storage. Microshoots from cold-stored encapsulated explants, which were rooted in solid MS nutrient medium with 0.5 μΜ of Indole-3-acetic acid (IAA) and transplanted to a substrate of peat-perlite (3:1, v/v), acclimatized successfully after application of 75 or 50% shading, which was gradually reduced, and were established with minimum losses in a greenhouse. For the genetic stability of the artificial seed-derived plantlets and compared with the mother plant, an assessment was conducted using Inter Simple Sequence Repeats (ISSRs) analysis. The ISSR profiles proved the genetic uniformity and clonal stability of the regenerated plantlets and their genetic resemblance to the mother plant. The present regeneration procedure could be used as an alternative method for the micropropagation of V. dentatum.

scholarly journals Conservation, Regeneration and Genetic Stability of Regenerants from Alginate-Encapsulated Shoot Explants of Gardenia jasminoides Ellis

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1666
Author(s):  
Stefanos Hatzilazarou ◽  
Stefanos Kostas ◽  
Theodora Nendou ◽  
Athanasios Economou
Keyword(s):  
Sodium Alginate ◽  
Calcium Chloride ◽  
Genetic Stability ◽  
Nutrient Medium ◽  
Shoot Tips ◽  
Mother Plant ◽  
Short Term ◽  
Alginate Encapsulation ◽  
Gardenia Jasminoides ◽  
Regeneration Response

The present study demonstrates the potential of the alginate encapsulation of shoot tips and nodal segments of Gardenia jasminoides Ellis, the short-term cold storage of artificial seeds and subsequent successful conversion to desirable, uniform and genetically stable plantlets. Shoot tips and first-node segments below them, derived from shoots of in vitro cultures, responded better than second-to-fourth-node segments on agar-solidified Murashige and Skoog (MS) nutrient medium and thus, they were used as explants for alginate encapsulation. Explant encapsulation in 2.5% sodium alginate in combination with 50 mM of calcium chloride resulted in the production of soft beads, while hardening in 100 mM of calcium chloride formed firm beads of uniform globular shape, suitable for handling. The addition of liquid MS nutrient medium in the sodium alginate solution doubled the subsequent germination response of the beads. The maintenance of alginate beads under light favored their germination response compared to maintenance in darkness. Encapsulated shoot tip explants of gardenia, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in their regeneration response (73.3, 68.9, 53.3%, respectively), whereas, non-encapsulated explants (naked), stored under the same time durations of cold conditions, exhibited a sharp decline in regeneration response up to entirely zeroing (48.9, 11.1, 0.0%, respectively). Shoots, derived from 12-week cold-stored encapsulated explants, were easily rooted in solid MS nutrient medium with the addition of 0.5 μM of Indole-3-acetic acid (IAA) and after transplantation of the rooted plantlets individually to pots containing a peat–perlite (3:1, v/v) substrate, they were successfully acclimatized in the greenhouse under the gradual reduction of 75 or 50% shading with survival rates of 95–100%. The genetic stability of the acclimatized plantlets was assessed and compared with the mother plant using inter simple sequence repeat (ISSR) markers. ISSR analysis confirmed that all regenerated plantlets were genetically identical to the mother plant. This procedure of artificial seed production could be useful for the short-term storage of germplasm and the production of genetically identical and stable plants as an alternative method of micropropagation in Gardenia jasminoides.

scholarly journals Peculiarities of in vitro parthenogenesis of unpollinated maize ovaries

2017 ◽  
Vol 6 (4) ◽  
pp. 10-13
Author(s):  
Tatyana Alekseevna Alatortseva
Keyword(s):  
Embryo Development ◽  
Nutrient Medium ◽  
Normal Development ◽  
Mother Plant ◽  
Maize Line ◽  
Mineral Components ◽  
Parthenogenetic Embryo ◽  
Direct Embryogenesis ◽  
Agricultural Plants

The maize line AT-1 is characterized by a hereditary predisposition to parthenogenesis. The aim of this investigation is to study parthenogenetic embryo development in the culture of unpollinated ovaries in vitro . The unpollinated ovaries were explanted in 1, 3, 5, 7, 10, 15 days after the appearance of stigmas from ears. The nutrient medium included mineral components of MS, vitamins, sucrose (9,0%), 2,4-D (2,0 mg/l), agar-agar. The structure of megagametophytes at the time of inoculation of the ovaries and on the 3rd, 7th, 14th, 21th, 28th day of cultivation was studied. The first divisions of unfertilized egg cells were observed on the 5th-7th day after appearance of stigmas from ears, independently from whether all this time the ovaries were on the mother plant or they were inoculated into the nutrient medium. The formation of the autonomous abnormal endosperm in some cultivated ovaries was detected. The abnormal endosperm disturbed normal development of the proembryo. As a rule, the ovaries with embryo and endosperm degenerated. In the absence of endosperm, the morphogenesis of parthenogenetic proembryos was carried out in one of two directions in vitro : 1) development of plants by direct embryogenesis; 2) regeneration of plants from numerous embryoids, raised on the surface of globular proembryos. The second direction was prevailed. The culture of unpollinated ovaries can be a promising method of mass haploid regenerants not only in maize, but also in other types of agricultural plants.

scholarly journals Assessment of Genetic Stability on In Vitro And Ex Vitro Plants of Ficus Carica Var. Black Jack Using Issr and Damd Markers

2021 ◽  
Author(s):  
Ankita Rajendra Parab ◽  
Chew Bee Lynn ◽  
Sreeramanan Subramaniam
Keyword(s):  
Stability Analysis ◽  
Genetic Stability ◽  
Similarity Index ◽  
Issr Markers ◽  
Ficus Carica ◽  
Ex Vitro ◽  
Regenerated Plants ◽  
Inter Simple Sequence Repeats ◽  
Dna Primers

Abstract In vitro propagation has been significant in producing a large number of genetically stable regenerated plants. Regenerated Ficus carica var. Black Jack plantlets were established using woody plant medium (WPM) supplemented with 20 µM 6-Benzylaminopurine (BAP) and 8 µM Indole-3-acetic acid (IAA) under different light treatments such as normal fluorescent white light (60 µmol.m− 2.s− 1), and four different LED spectra, white (400– 700nm), blue (440nm), red (660nm) and blue + red (440nm + 660nm). Genetic stability analysis was performed on the in vitro and ex vitro plants of Ficus carica var. Black Jack. Ten (10) primers of each ISSR and DAMD molecular marker were used to assess the genetic stability of the eight (8) samples of Ficus carica var. Black Jack, acquired over two years. The findings of this study revealed that inter simple sequence repeats (ISSR) and directed amplification of minisatellite DNA (DAMD) markers (DNA primers) are efficient in determining the polymorphism and monomorphism percentage among the in vitro and ex vitro samples of Ficus carica var. Black Jack. ISSR markers showed 97.87% of monomorphism whereas DAMD markers showed 100% monomorphism. Polymorphism of 2.13% was observed for the UBC840 ISSR – DNA primer which was negated under the genetic similarity index analysis for the eight samples. It is recommended that genetic stability analysis should be performed for long-term maintenance of micropropagated plants.

scholarly journals Genetic Stability of In vitro Multiplied Phalaenopsis gigantea Protocorm-like Bodies as Affected by Chitosan

2013 ◽  
Vol 41 (1) ◽  
pp. 177 ◽  
Author(s):  
Samira SAMARFARD ◽  
Mihdzar A. KADIR ◽  
Saleh B. KADZIMIN ◽  
Seyedali RAVANFAR ◽  
Halimi M. SAUD
Keyword(s):  
Plant Growth ◽  
Plant Species ◽  
Genetic Stability ◽  
Liquid Culture ◽  
Issr Markers ◽  
Mother Plant ◽  
Liquid Media ◽  
Carbohydrate Polymer ◽  
Media Types

Chitosan is a carbohydrate polymer derivative of chitin which presents in shell of crustaceans. This biopolymer is a non toxic and environmentally friendly, considered as a plant growth stimulator in some plant species. The present study investigates the effects of chitosan and media types on multiplication and genetic stability of Phalaenopsis gigantea protocorm-like bodies (PLBs). PLBs were inoculated in liquid New Dogashima Medium (NDM) and Vacin and Went (VW) supplemented with various concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L). The highest PLB multiplication was observed on VW and NDM supplemented with 10 mg/L chitosan with mean number of PLBs 177 and 147, respectively. Chitosan promoted the formation of juvenile leaves and the highest number was observed in NDM supplemented with 20 mg/L chitosan with mean number of 66 leaves after 8 weeks of culture. Genetic stability was assessed among mother plant and secondary PLBs after 2, 4, 6, and 8 weeks of culture in liquid media. 8 out of 10 ISSR markers produced a total of 275 clear and reproducible bands with mean of 6.9 bands per primer. The secondary PLBs produced during sub-culturing process of chitosan treated liquid culture were genetically uniform and similar to mother plant.

scholarly journals In Vitro Propagation and Detection of Somaclonal Variation in Phalaenopsis gigantea as Affected by Chitosan and Thidiazuron Combinations

HortScience ◽  
2014 ◽  
Vol 49 (1) ◽  
pp. 82-88 ◽  
Author(s):  
Samira Samarfard ◽  
Mihdzar A. Kadir ◽  
Saleh B. Kadzimin ◽  
Halimi M. Saud ◽  
Seyed Ali Ravanfar ◽  
...  
Keyword(s):  
Genetic Stability ◽  
In Vitro Propagation ◽  
Genetic Fidelity ◽  
Issr Markers ◽  
Similarity Coefficient ◽  
Mother Plant ◽  
Apical Region ◽  
Sequence Repeat ◽  
In Vitro Methods

Protocorm-like bodies (PLBs) multiplication is one of the most preferable in vitro methods to increase the number of orchids that produce very few seeds or seeds that are not able to germinate. In the present study the effects of chitosan and thidiazuron (TDZ) combinations on multiplication, differentiation, and genetic stability of Phalaenopsis gigantea PLBs were investigated using different media. Initial PLBs were cultured in solid New Dogashima (ND) medium and Vacin and Went (VW) medium supplemented with different concentrations of chitosan (0, 5, 10, 15, 20, and 25 mg·L–1) and TDZ (0, 0.1, and 0.5 mg·L–1). The highest mean number of PLBs (353 PLBs) was observed in ND medium with 10 mg·L–1 chitosan and 0.1 mg·L–1 TDZ combination after 20 weeks of culture. Some PLBs differentiated into mature PLBs with a profusion of leaves on the apical region, and tiny plantlets started to develop after 10 weeks of culture. The highest mean number of shoots was observed in VW supplemented with 10 mg·L–1 chitosan and 0.5 mg·L–1 TDZ (16 shoots). Intersimple sequence repeat (ISSR) markers were used to determine the genetic fidelity among mother plant and PLBs obtained from each subculture stage of solid ND medium supplemented with 10 mg·L–1 chitosan and 0.1 mg·L–1 TDZ (the optimal treatment for PLB proliferation). Dissimilarity of 5% occurred between the mother plant and PLBs obtained after 16 weeks of culture. The range in the similarity coefficient varied from 0.80 to 1.0, and only 20% dissimilarity occurred between mother plant and PLBs after 20 weeks of culture.

Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

Comparative Investigation on Formation and Accumulation of Rare Phenylpropanoids in Plants and in vitro Cultures of Pimpinella major

1990 ◽  
Vol 45 (6) ◽  
pp. 602-606 ◽  
Author(s):  
B. Merkel ◽  
J. Reichling
Keyword(s):  
Callus Culture ◽  
Nutrient Medium ◽  
Callus Cultures ◽  
Comparative Investigation ◽  
In Vitro Cultures ◽  
Liquid Nutrient Medium ◽  
Whole Plants ◽  
Plant Seedlings

Abstract Unorganized callus and leaf/root-differentiating callus cultures of Pimpinella major have been established in liquid nutrient medium. Their capacity to accumulate rare phenylpropanoids such as epoxy-pseudoisoeugenol tiglate, epoxy-anol tiglate and anol tiglate was compared with that of seedlings and whole plants. The unorganized callus cultures were not able to accumulate any phenylpropanoids. In comparison, the leaf/root-differentiating callus culture promoted the accumulation of epoxy-pseudoisoeugenol tiglate (up to 90 mg/100 g fr.wt.) but not that of anol-derivatives. The accumulated amount of EPT in PMD-SH was comparable with that in plant seedlings.

scholarly journals Electrochemotherapy with Calcium Chloride and 17β-Estradiol Modulated Viability and Apoptosis Pathway in Human Ovarian Cancer

Pharmaceutics ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 19
Author(s):  
Zofia Łapińska ◽  
Michał Dębiński ◽  
Anna Szewczyk ◽  
Anna Choromańska ◽  
Julita Kulbacka ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Calcium Chloride ◽  
Morphological Changes ◽  
Cell Membrane Permeability ◽  
Immunocytochemical Staining ◽  
Ovarian Cancer Cells ◽  
Apoptosis Pathway ◽  
17Β Estradiol

Estrogens (Es) play a significant role in the carcinogenesis and progression of ovarian malignancies. Depending on the concentration, Es may have a protective or toxic effect on cells. Moreover, they can directly or indirectly affect the activity of membrane ion channels. In the presented study, we investigated in vitro the effectiveness of the ovarian cancer cells (MDAH-2774) pre-incubation with 17β-estradiol (E2; 10 µM) in the conventional chemotherapy (CT) and electrochemotherapy (ECT) with cisplatin or calcium chloride. We used three different protocols of electroporation including microseconds (µsEP) and nanoseconds (nsEP) range. The cytotoxic effect of the applied treatment was examined by the MTT assay. We used fluorescent staining and holotomographic imaging to observe morphological changes. The immunocytochemical staining evaluated the expression of the caspase-12. The electroporation process’s effectiveness was analyzed by a flow cytometer using the Yo-Pro™-1 dye absorption assay. We found that pre-incubation of ovarian cancer cells with 17β-estradiol may effectively enhance the chemo- and electrochemotherapy with cisplatin and calcium chloride. At the same time, estradiol reduced the effectiveness of electroporation, which may indicate that the mechanism of increasing the effectiveness of ECT by E2 is not related to the change of cell membrane permeability.

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