scholarly journals Optimization of protoplast regeneration in the model plant Arabidopsis thaliana

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yeong Yeop Jeong ◽  
Hun-Young Lee ◽  
Suk Weon Kim ◽  
Yoo-Sun Noh ◽  
Pil Joon Seo
Keyword(s):  
Tissue Culture ◽  
Arabidopsis Thaliana ◽  
Plant Regeneration ◽  
Tissue Regeneration ◽  
De Novo ◽  
Callus Formation ◽  
Protoplast Regeneration ◽  
Culture Methods ◽  
Cell And Tissue Culture

Abstract Background Plants have a remarkable reprogramming potential, which facilitates plant regeneration, especially from a single cell. Protoplasts have the ability to form a cell wall and undergo cell division, allowing whole plant regeneration. With the growing need for protoplast regeneration in genetic engineering and genome editing, fundamental studies that enhance our understanding of cell cycle re-entry, pluripotency acquisition, and de novo tissue regeneration are essential. To conduct these studies, a reproducible and efficient protoplast regeneration method using model plants is necessary. Results Here, we optimized cell and tissue culture methods for improving protoplast regeneration efficiency in Arabidopsis thaliana. Protoplasts were isolated from whole seedlings of four different Arabidopsis ecotypes including Columbia (Col-0), Wassilewskija (Ws-2), Nossen (No-0), and HR (HR-10). Among these ecotypes, Ws-2 showed the highest potential for protoplast regeneration. A modified thin alginate layer was applied to the protoplast culture at an optimal density of 1 × 106 protoplasts/mL. Following callus formation and de novo shoot regeneration, the regenerated inflorescence stems were used for de novo root organogenesis. The entire protoplast regeneration process was completed within 15 weeks. The in vitro regenerated plants were fertile and produced morphologically normal progenies. Conclusion The cell and tissue culture system optimized in this study for protoplast regeneration is efficient and reproducible. This method of Arabidopsis protoplast regeneration can be used for fundamental studies on pluripotency establishment and de novo tissue regeneration.

scholarly journals Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum

2021 ◽  
Author(s):  
Ping Che ◽  
Emily Wu ◽  
Marissa K. Simon ◽  
Ajith Anand ◽  
Keith Lowe ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Genome Editing ◽  
De Novo ◽  
Callus Formation ◽  
Fold Increase ◽  
Control Treatment ◽  
In Planta ◽  
Culture Methods ◽  
Agrobacterium Mediated Transformation ◽  
Sorghum Genotypes

For many important crops including sorghum, the use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but more critically, by the ability to regenerate a fertile plant from the engineered cell through conventional tissue culture methods. Here, we report that Wuschel2 (Wus2)-enabled sorghum (Sorghum bicolor L.) transformation increases not only the efficiency of transgenic plant recovery, but also increases the observed efficiency of CRISPR/Cas-targeted genome editing within the population of T0 plants. Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased regeneration capacity, resulting in higher transformation efficiency across different sorghum genotypes. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrated up to an additional 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled, Agrobacterium-mediated transformation (compared to the control treatment with no Wus2), across various targeted loci in different sorghum genotypes. Wus2-enabled genome editing may be applicable to other crops and increasingly popular in planta transformation strategies.

scholarly journals Rejuvenation of chicory and lettuce plants following phase change in tissue culture

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Anthony J. Conner ◽  
Helen Searle ◽  
Jeanne M. E. Jacobs
Keyword(s):  
Tissue Culture ◽  
Shoot Regeneration ◽  
Seed Set ◽  
Genetic Manipulation ◽  
Adventitious Shoot ◽  
In Vitro Flowering ◽  
Phase Changes ◽  
Adventitious Shoot Regeneration ◽  
Cell And Tissue Culture

Abstract Background A frequent problem associated with the tissue culture of Compositae species such as chicory (Cichorium intybus L.) and lettuce (Lactuca sativa L.) is the premature bolting to in vitro flowering of regenerated plants. Plants exhibiting such phase changes have poor survival and poor seed set upon transfer from tissue culture to greenhouse conditions. This can result in the loss of valuable plant lines following applications of cell and tissue culture for genetic manipulation. Results This study demonstrates that chicory and lettuce plants exhibiting stable in vitro flowering can be rejuvenated by a further cycle of adventitious shoot regeneration from cauline leaves. The resulting rejuvenated plants exhibit substantially improved performance following transfer to greenhouse conditions, with increased frequency of plant survival, a doubling of the frequency of plants that flowered, and substantially increased seed production. Conclusion As soon as in vitro flowering is observed in unique highly-valued chicory and lettuce lines, a further cycle of adventitious shoot regeneration from cauline leaves should be implemented to induce rejuvenation. This re-establishes a juvenile phase accompanied by in vitro rosette formation, resulting in substantially improved survival, flowering and seed set in a greenhouse, thereby ensuring the recovery of future generations from lines genetically manipulated in cell and tissue culture.

Tests by Tissue Culture Methods on the Nature of Immunity Transplanted Skin

1948 ◽  
Vol s3-89 (7) ◽  
pp. 239-252
Author(s):  
P. B. MEDAWAR
Keyword(s):  
Tissue Culture ◽  
Cell Division ◽  
Epidermal Cell ◽  
Cell Suspensions ◽  
Acquired Immunity ◽  
Culture Methods ◽  
Division Frequency

The transplantation of skin from one rabbit to another elicits a reaction that conforms in main outline with that of an actively acquired immunity. The experiments described in this paper were designed to test the hypothesis that the regression of such grafts is secured by the action of antibodies demonstrable in vitro. Skin from adult rabbits has therefore been cultivated in the presence of serum and growing mesenchymal tissues derived solely from rabbits heavily and specifically immunized against it. Immune sera and tissues are without effect on the survival, cell-division frequency and migratory activities of explanted skin, and agglutinins for epidermal cell suspensions are not demonstrable in immune sera. With certain stated qualifications, it has therefore been concluded that the occurrence of free antibodies is not a sufficient explanation of the regression of skin homografts in vivo.

scholarly journals Phloroglucinol Mediated Plant Regeneration of Ornithogalum dubium as the Sole “Hormone-Like Supplement” in Plant Tissue Culture Long-Term Experiments

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 929
Author(s):  
Carloalberto Petti
Keyword(s):  
Tissue Culture ◽  
Plant Regeneration ◽  
Callus Formation ◽  
Total Phenolic ◽  
Naturally Occurring ◽  
Long Term Experiments ◽  
Direct Embryogenesis ◽  
The Media ◽  
Synthetic Hormones

Tissue culture is an essential requirement in plant science to preserve genetic resources and to expand naturally occurring germplasm. A variety of naturally occurring and synthetic hormones are available to induce the processes of dedifferentiation and redifferentiation. Not all plant material is susceptible to tissue culture, and often complex media and hormone requirements are needed to achieve successful plant propagations. The availability of new hormones or chemicals acting as hormones are critical to the expansion of tissue culture potentials. Phloroglucinol has been shown to have certain hormone-like properties in a variety of studies. Ornithogalum dubium, an important geophyte species, was used to characterise the potential of phloroglucinol as the sole plant-like hormone in a tissue culture experiment. Tissue culture, plant regeneration, total phenolic and genetic variability were established by applying a variety of methods throughout long-term experiments. Phloroglucinol did induce callus formation and plant regeneration when used as the sole supplement in the media at a rate of 37%, thus demonstrating auxin/cytokines-like properties. Callus formation was of 3 types, friable and cellular, hard and compact, and a mixture of the two. The important finding was that direct somatogenesis did occur albeit more frequently on younger tissue, whereby rates of induction were up to 52%. It is concluded that phloroglucinol acts as a “hormone-like” molecule and can trigger direct embryogenesis without callus formation.

Control of morphogenetic pathways in thin cell layers of tobacco by pH

1989 ◽  
Vol 67 (3) ◽  
pp. 650-654 ◽  
Author(s):  
A. Cousson ◽  
P. Toubart ◽  
K. Tran Thanh Van
Keyword(s):  
Butyric Acid ◽  
De Novo ◽  
Culture Media ◽  
Callus Formation ◽  
Medium Ph ◽  
Vegetative Buds ◽  
Cell Layers ◽  
Initial Medium ◽  
Vegetative Bud

Thin cell layer explants of tobacco were floated in vitro on the surface of liquid culture media. The initial exogenous concentrations of indolyl-3-butyric acid, and kinetin, the initial medium pH, and the explant density were varied. Various patterns of de novo and direct differentiation without any intermediate callus (flower, vegetative bud, root) as well as the absence of morphogenesis and callus formation without any subsequent organogenesis were separately controlled on 100% of the explants. On the same exogenous combination of glucose, indolyl-3-butyric acid, and kinetin, changes in initial medium pH changed the pattern of morphogenesis. For a given initial exogenous indolyl-3-butyric acid concentration, vegetative buds were obtained at either pH 6.1 or 7.8, whereas a mixture of flowers and vegetative buds was obtained at pH 6.8. Furthermore, changes in explant density changed the morphogenetic response. It is suggested that the effects of the initial medium pH and explant density on morphogenesis may be related partially to modifications of the physicochemical properties of the cell wall and (or) plasmalemma.

Small-scale cultivation of cells and tissues from gobiid teleosts

1975 ◽  
Vol 55 (4) ◽  
pp. 933-938
Author(s):  
C. J. Webb
Keyword(s):  
Tissue Culture ◽  
Coastal Ecosystems ◽  
Estuarine Fish ◽  
Small Scale ◽  
Freshwater Teleost ◽  
Fish Tissues ◽  
Cell And Tissue Culture

Although freshwater teleost cell and tissue culture is an established field, the in vitro maintenance and growth of marine or estuarine fish tissues and cells is less well reported (Wolf & Quimby, 1969). Small demersal gobiid teleosts of the genus Pomatoschistus Gill, 1864 are widely distributed and very abundant around the coast of Britain and play an extremely important role in estuarine and coastal ecosystems (Hartley, 1940; Miller, 1963, Ph.D. Thesis, University of Liverpool; Green, 1968). Excluding the in vitro maintenance of whole organs (e.g. Bonnin, 1971a, b and Doneen & Bern, 1974) the culture of cells or tissues from gobiids has not been reported.

Study on Tissue Culture of Sweet Broad Pea

2014 ◽  
Vol 644-650 ◽  
pp. 5407-5410
Author(s):  
Hui Fang Chi
Keyword(s):  
Tissue Culture ◽  
Plant Regeneration ◽  
Culture Medium ◽  
Callus Formation ◽  
Formation Rate ◽  
Adventitious Bud ◽  
Experimental Basis ◽  
Research Results

s. The cotyledons, Internodes, leaves and stems of sweet broad pea were studied on tissue culture. Research results show that: The ability of different explants for callus formation and adventitious bud differentiation in different culture medium is different. The callus formation rate and sprouting rate of Internodes is significantly higher than other explants, which is a ideal material for tissue culture. The callus formation rate of Internodes was 100% in MS +BA1.0 mg/L+NAA 1.0 mg/L and MS+ 2, 4-D 0.5 mg/L; The bud differentiation is best at the medium of MS+ 6-BA 2 mg/L, which reached 86.7%; the rooting rate was 83.3% at the medium of MS+ NAA 3mg/L. The study provides a experimental basis for further study on the plant regeneration in the sweet broad pea.

scholarly journals 664 PB 104 AGROBACTERIUM INFECTION OF WHITE ASH (FRAXINUS AMERICANA L.)

HortScience ◽  
1994 ◽  
Vol 29 (5) ◽  
pp. 528a-528
Author(s):  
Sharon A. Bates ◽  
John E. Preece ◽  
John H. Yopp
Keyword(s):  
Tissue Culture ◽  
Plant Growth ◽  
Growth Regulators ◽  
Callus Formation ◽  
High Humidity ◽  
Ms Medium ◽  
Gall Formation ◽  
White Ash ◽  
Seedling Explants

Both greenhouse-grown white ash plants derived from tissue culture and rooted microshoots in high humidity trays were inoculated with 11 tumor-inducing Agrobacterium strains. Eight strains stimulated mutative gall formation. Plants inoculated with strain A281 exhibited a higher frequency of callus formation (greenhouse-22.2%; microshoots-18.8%) than other strains at the site of the wound. Therefore, strain A281 was used to inoculate seed and seedling explants in vitro. Explants were placed on MS medium containiner no plant growth regulators and inoculated at 0, 3, 5, 7, or 10 days after initiation. Plants inoculated at 10 days showed a higher frequency of callus formation (16.4%) than with earlier inoculations. Also, rewounding of the explant at inoculation resulted in a higher frequency of callus formation (11.3%) compared to not rewounding the explant (3.9%).

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