Genetic Engineering and In Vitro Manipulation of Plant Cells — Technical Advances

Author(s):  
Y. P. S. Bajaj
2021 ◽  
Vol 12 ◽  
Author(s):  
Constanca Figueiredo ◽  
Rainer Blasczyk

Patelet transfusion refractoriness remains a relevant hurdle in the treatment of severe alloimmunized thrombocytopenic patients. Antibodies specific for the human leukocyte antigens (HLA) class I are considered the major immunological cause for PLT transfusion refractoriness. Due to the insufficient availability of HLA-matched PLTs, the development of new technologies is highly desirable to provide an adequate management of thrombocytopenia in immunized patients. Blood pharming is a promising strategy not only to generate an alternative to donor blood products, but it may offer the possibility to optimize the therapeutic effect of the produced blood cells by genetic modification. Recently, enormous technical advances in the field of in vitro production of megakaryocytes (MKs) and PLTs have been achieved by combining progresses made at different levels including identification of suitable cell sources, cell pharming technologies, bioreactors and application of genetic engineering tools. In particular, use of RNA interference, TALEN and CRISPR/Cas9 nucleases or nickases has allowed for the generation of HLA universal PLTs with the potential to survive under refractoriness conditions. Genetically engineered HLA-silenced MKs and PLTs were shown to be functional and to have the capability to survive cell- and antibody-mediated cytotoxicity using in vitro and in vivo models. This review is focused on the methods to generate in vitro genetically engineered MKs and PLTs with the capacity to evade allogeneic immune responses.


1991 ◽  
Vol 69 (4) ◽  
pp. 822-830 ◽  
Author(s):  
Ulisses G. Batista ◽  
Verna J. Higgins

The production and distribution of the phytoalexin falcarindiol in tomato foliage infected with leaf mold was examined to determine how the fungus Cladosporium fulvum is able to colonize and sporulate in an apparently antifungal environment. In a compatible interaction (cv. Potentate – C. fulvum race 2.3), by 12 and 15 days after inoculation, solvent-extractable falcarindiol and two other phytoalexins from tomato, compound 2 (probably falcarinol) and compound 3 (unidentified), reached concentrations considerably in excess of ED50 values for inhibition of the fungus. In contrast, intercellular (apoplastic) fluids obtained from similarly infected leaflets contained only traces of falcarindiol. ED50 values for germination and germ-tube growth of C. fulvum increased as the incubation time was extended, suggesting that adaptation or recovery was possible at the concentrations tested. In in vitro experiments, C. fulvum appeared to readily metabolize falcarindiol, as did a Lycopersicon cell suspension culture. Binding of falcarindiol to living and dead fungal and plant cells was also observed. Falcarindiol, injected into tomato leaflets, decreased rapidly and was only recovered in trace amounts by 24 h. The results suggest that falcarindiol and probably the two other phytoalexins do not reach sufficient concentrations in the apoplast of an infected susceptible leaf to have an effect on growth and sporulation of C. fulvum. Key words: leaf mold, Fulvia fulva, falcarindiol, falcarinol.


1988 ◽  
Vol 252 (1) ◽  
pp. 39-45 ◽  
Author(s):  
S A Elhussein ◽  
J A Miernyk ◽  
J B Ohlrogge

1. An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme. 2. Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography. 3. The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively. Synthase activity was inhibited in vitro by the reaction product 3′,5′-ADP. 4. Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.


1994 ◽  
Vol 107 (8) ◽  
pp. 2249-2257 ◽  
Author(s):  
T. Asada ◽  
H. Shibaoka

As part of our efforts to understand the molecular basis of the microtubule-associated motility that is involved in cytokinesis in higher plant cells, an attempt was made to identify proteins with the ability to translocate microtubules in an extract from isolated phragmoplasts. Homogenization of isolated phragmoplasts in a solution that contained MgATP, MgGTP and a high concentration of NaCl resulted in the release from phragmoplasts of factors with ATPase and GTPase activity that were stimulated by microtubules. A protein fraction with microtubule-dependent ATPase and GTPase activity caused minus-end-headed gliding of microtubules in the presence of ATP or GTP. Polypeptides with microtubule-translocating activity cosedimented with microtubules that had been assembled in vitro from brain tubulin and were dissociated from sedimented microtubules by addition of ATP or GTP. After cosedimentation and dissociation procedures, a 125 kDa polypeptide and a 120 kDa polypeptide were recovered in a fraction that supported minus-end-headed gliding of microtubules. The rate of microtubule gliding that was caused by the fraction that contained the 125 kDa and 120 kDa polypeptides as main components was 1.28 microns/minute in the presence of ATP and 0.50 microns/minute in the presence of GTP. This fraction contained some microtubule-associated polypeptides in addition to the 125 kDa and 120 kDa polypeptides, but a fraction that contained only these additional polypeptides did not cause any translocation of microtubules. Thus, it appeared that the 125 kDa and 120 kDa polypeptides were responsible for translocation of microtubules. These polypeptides with plus-end-directed motor activity may play an important role in formation of the cell plate and in the organization of the phragmoplast.


1999 ◽  
Vol 155 (3) ◽  
pp. 376-386 ◽  
Author(s):  
Linda K. Adams ◽  
Erica E. Benson ◽  
Harry J. Staines ◽  
David H. Bremner ◽  
Stephen Millam ◽  
...  

Materials ◽  
2021 ◽  
Vol 14 (18) ◽  
pp. 5340
Author(s):  
Alicja Tymoszuk

The interactions between nanoparticles and plant cells are still not sufficiently understood, and studies related to this subject are of scientific and practical importance. Silver nanoparticles (AgNPs) are one of the most commonly produced and used nanomaterials. This study aimed to investigate the influence of AgNPs applied at the concentrations of 0, 50, and 100 mg·L−1 during the process of in vitro germination as well as the biometric and biochemical parameters of developed seedlings in three vegetable species: Solanum lycopersicum L. ‘Poranek’, Raphanus sativus L. var. sativus ‘Ramona’, and Brassica oleracea var. sabellica ‘Nero di Toscana’. The application of AgNPs did not affect the germination efficiency; however, diverse results were reported for the growth and biochemical activity of the seedlings, depending on the species tested and the AgNPs concentration. Tomato seedlings treated with nanoparticles, particularly at 100 mg·L−1, had shorter shoots with lower fresh and dry weights and produced roots with lower fresh weight. Simultaneously, at the biochemical level, a decrease in the content of chlorophylls and carotenoids and an increase in the anthocyanins content and guaiacol peroxidase (GPOX) activity were reported. AgNPs-treated radish plants had shorter shoots of higher fresh and dry weight and longer roots with lower fresh weight. Treatment with 50 mg·L−1 and 100 mg·L−1 resulted in the highest and lowest accumulation of chlorophylls and carotenoids in the leaves, respectively; however, seedlings treated with 100 mg·L−1 produced less anthocyanins and polyphenols and exhibited lower GPOX activity. In kale, AgNPs-derived seedlings had a lower content of chlorophylls, carotenoids, and anthocyanins but higher GPOX activity of and were characterized by higher fresh and dry shoot weights and higher heterogeneous biometric parameters of the roots. The results of these experiments may be of great significance for broadening the scope of knowledge on the influence of AgNPs on plant cells and the micropropagation of the vegetable species. Future studies should be aimed at testing lower or even higher concentrations of AgNPs and other NPs and to evaluate the genetic stability of NPs-treated vegetable crops and their yielding efficiency.


Ceiba ◽  
2016 ◽  
Vol 54 (1) ◽  
pp. 72-81
Author(s):  
Alison L. Van Eenennaam

Biotechnologies have been an integral part of improvements in animal genetics, nutrition and health over the past century. Many biotechnologies have become fundamental components of efficient livestock production systems. The genetic improvements that have been enabled by biotechnologies have dramatically decreased the environmental footprint of animal protein production in many parts of the world, and continued innovation is required to address the projected increase in demand for animal products in the future. Breeding programs increasingly utilize a combination of advanced reproductive technologies and genomic tools to accelerate the rate of genetic gain by manipulating components of the breeder’s equation. The use of these biotechnologies and breeding methods has met with little public opposition. In contrast, the use of modern biotechnologies, defined as those that employ the use of in vitro nucleic acid techniques, have been highly controversial, especially those involving the use of genetic engineering. This modern biotechnology distinction is somewhat arbitrary as there are a number of biotechnologies that involve the use of in vitro processes, and many result in genetic modifications that are indistinguishable from the naturally-occurring variation that is the driver of both traditional breeding programs and evolution. A number of useful traits including disease resistance and animal welfare traits have been successfully introduced into various livestock species using both genetic engineering and gene editing techniques. Ultimately these techniques complement the genetic improvement that can be accomplished using traditional selection techniques and, if judged acceptable, offer an opportunity to synergistically accelerate genetic improvement in food animal species.


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