In vitro somatic embryogenesis in callus cultures ofAzadirachta indicaA. Juss.—a multipurpose tree

2005 ◽  
Vol 10 (4) ◽  
pp. 263-267 ◽  
Author(s):  
Gyana Ranjan Rout
Keyword(s):  
Somatic Embryogenesis ◽  
Callus Cultures ◽  
Multipurpose Tree

Induction of somatic embryogenesis in Siberian spruce (Picea obovata) in in vitro culture

2021 ◽  
pp. 6-20
Author(s):  
Iraida N. Tretyakova ◽  
◽  
Maria E. Park ◽  
Angelica P. Pakhomova ◽  
Irina S. Sheveleva ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
In Vitro Culture ◽  
Cell Lines ◽  
Nutrient Medium ◽  
Somatic Embryos ◽  
Callus Cultures ◽  
Zygotic Embryos ◽  
Globular Embryo ◽  
Siberian Spruce

The biotechnology of somatic embryogenesis in in vitro culture is the most promising direction in the reproduction of conifers. The use of this technology makes it possible not only to massively propagate the best genotypes of trees, but also serves a model for studying the structural, physiological and molecular and genetic mechanisms of both somatic and zygotic embryogenesis in conifers. The main aim of this research was to obtain embryogenic cultures (ECs) producing somatic embryos and embryonic suspension mass (ESM) of Picea obovata. The studies were carried out in 2014-2019 on 30 Siberian spruce trees growing in the vicinity of the city of Krasnoyarsk. To detect genotypes competent for somatic embryogenesis, new donor trees were selected every year for the experiment. 3-10 cones were collected from each tree at different stages of embryo development: globular embryo (the first decade of July), the initiation stage cotyledons (second decade of July), the stage of developed cotyledons (third decade of July) and mature embryos (August). Sterilized explants (zygotic embryos at different stages of development) were introduced into in vitro culture on basic media DCR (Gupta PK and Durzan DJ, 1985), ½LV (Litvay JD et al., 1985), MS (Murashige T and Skoog F, 1962) and AI (Tretyakova IN, 2012). All media were supplemented with myo-inositol - 100 mg/L, casein hydrolyzate - 500-1000 mg/L, L-glutamine - 500 mg/L, sucrose - 30 g/L and agar - 7 g/L. Ascorbic acid at a concentration of 400 mg/L was used as an antioxidant. The level of growth regulators was: 2,4-dichlorophenoxyacetic acid (2,4-D) - 2 mg/L and N6 -benzoaminopurine (BAP) - 1 mg/L. For the proliferation of the ESM, DCR and AI basic media containing 2,4-D (2 mg/L), BAP (0.5 mg/L) and sucrose (20 g/L) were used. The pH was adjusted to pH = 5.8. All culture medium and components were sterilized depending on their termolabile properties. Under aseptic conditions, embryos were removed from megagametophytes and inoculated into nutrient media, 10 explants per flask in 25 replicates. The cultures were incubated in the dark at 24 ± 1 °C. Subcultivation to fresh nutrient medium was carried out every 14 days. To control the quality of cell lines (CL) during subculturing, we performed cytological analyzes using temporary preparations (3-5 preparations for each CL). We evaluated the quality of the embryogenicity of the cultures by the presence of even single structures with pronounced polarity - a globular embryo with a suspensor. The results of the study showed that the induction of callus cultures of Siberian spruce is influenced by such factors as the development stage of the explant, the nutrient medium and the genotype of the donor tree. The introduction of P. obovata immature zygotic embryos into in vitro culture at the stage of the globular embryo, both with megagametophytes and extracted from them, turned out to be ineffective. The induction of callus cultures in Siberian spruce was significantly reduced when mature zygotic embryos were introduced into the culture in vitro. The highest response of explants of Siberian spruce was at the stage of developed cotyledons (See Table 1). In the DCR medium, 90% of explants formed callus (See Table 2). The mineral composition of the media did not significantly affect the induction of callus formation in Siberian spruce. The exception was the MS medium, in which callus cultures were formed only in 41% of explants (See Table 2). The growth of callus cultures was most active in the DCR medium. After 6 months of cultivation, 15-32% of calli remained viable (See Table 2). Cytological analysis of callus cultures showed that they include cells of different types (See Fig. 1 and 2). The first type of cells consisted of elongated cells reaching a length of 10 ± 3 μm, others consisted of isodiametric cells with a diameter of 60 ± 3.5 μm. The somatic embryo globule and embryonic tubes were formed from elongated cells. Isodiametric cells were actively dividing and forming callus. Only 3 cell lines (out of 300 cell lines) belonging to two donor trees had an active ability to proliferate. Globular somatic embryos were actively forming in these cell lines (See Fig. 3). An actively proliferating ESM was formed. Thus, we carried out a comprehensive assessment of the factors influencing the induction of somatic embryogenesis in Siberian spruce. The results obtained indicate that for the successful formation of somatic embryos, the determining factor is not only the choice of donor plants, but also the development stage of the explant. We found that the best stage in the development of zygotic embryos when introduced into in vitro culture of Siberian spruce is the stage of immature embryos with formed cotyledons, while the DCR, ½LV and AI nutrient medium supplemented with growth regulators (2.4-D and BAP) is optimal.

Somatic Embryogenesis and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

1970 ◽  
Vol 19 (1) ◽  
pp. 89-99
Author(s):  
K. Choudhary ◽  
M. Singh ◽  
M. S. Rathore ◽  
N. S. Shekhawat
Keyword(s):  
Somatic Embryogenesis ◽  
Growth Regulators ◽  
Somatic Embryos ◽  
Basal Medium ◽  
Long Term Study ◽  
Continuous Application ◽  
First Time ◽  
Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l  of 2,4-D and 0.5 mg/l  of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture  legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

In Vitro Establishment of Powdery Mildew (Microsphaera pulchra) on Microshoots of Flowering Dogwood

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 506a-506
Author(s):  
L.A. Klein ◽  
M.T. Windham ◽  
R.N. Trigiano
Keyword(s):  
Powdery Mildew ◽  
Woody Plant ◽  
Infected Individual ◽  
Callus Cultures ◽  
Fungal Culture ◽  
Culture Collections ◽  
Flowering Dogwood ◽  
Plant Parasite ◽  
In Vitro Establishment

Microshoot and callus cultures of Cornus florida (flowering dogwood), which were grown on woody plant medium amended with BA, were inoculated with Microsphaera pulchra (an obligate plant parasite) by gently shaking infected leaves bearing numerous conidia over the tissue. Culture dishes were sealed with parafilm and incubated at 24 °C with 25 mol·m–2·s–1 provided by cool fluorescent bulbs for 15 h. Cultures were examined with a dissecting scope every 24 h and cultures transferred when contaminating fungi were present. Specimens were prepared light microscopy and SEM. The fungus infected individual callus cells, but did not sporulate. In contrast, powdery mildew was well-established (both primary and secondary hyphae) in 70% of the microshoot cultures after 6 days and sporulated on 20% by 7 to 8 days. The cellular relationship between host and pathogen in vitro was similar to that found in greenhouse-grown plants. This technique has possible applications in maintaining fungal culture collections and studying host–pathogen relationships under more stringently controlled conditions.

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 Telomere Length in Norway Spruce during Somatic Embryogenesis and Cryopreservation

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 416
Author(s):  
Tuija Aronen ◽  
Susanna Virta ◽  
Saila Varis
Keyword(s):  
Somatic Embryogenesis ◽  
Telomere Length ◽  
Norway Spruce ◽  
Genotypic Variation ◽  
Production Capacity ◽  
Stress Factors ◽  
Embryo Production ◽  
One Year ◽  
Plant Telomeres

Telomeres i.e., termini of the eukaryotic chromosomes protect chromosomes during DNA replication. Shortening of telomeres, either due to stress or ageing is related to replicative cellular senescence. There is little information on the effect of biotechnological methods, such as tissue culture via somatic embryogenesis (SE) or cryopreservation on plant telomeres, even if these techniques are widely applied. The aim of the present study was to examine telomeres of Norway spruce (Picea abies (L.) Karst.) during SE initiation, proliferation, embryo maturation, and cryopreservation to reveal potential ageing or stress-related effects that could explain variation observed at SE process. Altogether, 33 genotypes from 25 families were studied. SE initiation containing several stress factors cause telomere shortening in Norway spruce. Following initiation, the telomere length of the embryogenic tissues (ETs) and embryos produced remains unchanged up to one year of culture, with remarkable genotypic variation. Being prolonged in vitro culture can, however, shorten the telomeres and should be avoided. This is achieved by successful cryopreservation treatment preserving telomere length. Somatic embryo production capacity of the ETs was observed to vary a lot not only among the genotypes, but also from one timepoint to another. No connection between embryo production and telomere length was found, so this variation remains unexplained.

scholarly journals Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Renata Orłowska
Keyword(s):  
Tissue Culture ◽  
Somatic Embryogenesis ◽  
Taguchi Method ◽  
Culture Conditions ◽  
Dna Demethylation ◽  
Silver Ion ◽  
Donor Plant ◽  
In Vitro Tissue Culture ◽  
Induced Variation

Abstract Background Somatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. The DNA changes may be the outcome of induction media ingredients (i.e., copper and silver ions) and their concentrations and time of in vitro cultures. Results This study optimised the level of copper and silver ion concentration in culture media parallel with the induction medium longevity step towards obtaining barley regenerants via somatic embryogenesis with a minimum or maximum level of tissue culture-induced differences between the donor plant and its regenerants. The optimisation process is based on tissue culture-induced variation evaluated via the metAFLP approach for regenerants derived under varying in vitro tissue culture conditions and exploited by the Taguchi method. In the optimisation and verification experiments, various copper and silver ion concentrations and the different number of days differentiated the tested trials concerning the tissue culture-induced variation level, DNA demethylation, and de novo methylation, including symmetric (CG, CHG) and asymmetric (CHH) DNA sequence contexts. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful. The main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context. Conclusions The combination of tissue culture-induced variation evaluated for eight experimental trials and implementation of the Taguchi method allowed the optimisation of the in vitro tissue culture conditions towards the minimum and maximum differences between a source of tissue explants (donor plant) and its regenerants from somatic embryos. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context.

scholarly journals In vitro metabolism of Tebuconazole, Flurtamone, Fenhexamid, Metalaxyl‐M and Spirodiclofen in Cannabis sativa L. (hemp) callus cultures

2021 ◽  
Author(s):  
Leonie Hillebrands ◽  
Marc Lamshoeft ◽  
Andreas Lagojda ◽  
Andreas Stork ◽  
Oliver Kayser
Keyword(s):  
Cannabis Sativa ◽  
Callus Cultures ◽  
In Vitro Metabolism
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