We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.

https://www.pnas.org/content/pnas/82/17/5824.full.pdf

Expression of genes transferred into monocot and dicot plant cells by electroporation

1 Dielectrophoresis and Rotation of Cells.- 2 Cellular Spin Resonance.- 3 Dielectrophoresis: Behavior of Microorganisms and Effect of Electric Fields on Orientation Phenomena.- 4 The Relaxation Hysteresis of Membrane Electroporation.- 5 Electrical Breakdown of Lipid Bilayer Membranes: Phenomenology and Mechanism.- 6 Stochastic Model of Electric Field-Induced Membrane Pores.- 7 Theory of Electroporation.- 8 Leaks Induced by Electrical Breakdown in the Erythrocyte Membrane.- 9 Electroporation of Cell Membranes: Mechanisms and Applications.- 10 Electrofusion Kinetics: Studies Using Electron Microscopy and Fluorescence Contents Mixing.- 11 Electrofusion of Lipid Bilayers.- 12 Role of Proteases in Electrofusion of Mammalian Cells.- 13 Electrofusion of Mammalian Cells and Giant Unilamellar Vesicles.- 14 Cell Fusion and Cell Poration by Pulsed Radio-Frequency Electric Fields.- 15 The Mechanism of Electroporation and Electrofusion in Erythrocyte Membranes.- 16 Producing Monoclonal Antibodies by Electrofusion.- 17 Generation of Human Hybridomas by Electrofusion.- 18 Gaining Access to the Cytosol: Clues to the Control and Mechanisms of Exocytosis and Signal Transduction Coupling.- 19 Gene Transfer by Electroporation: A Practical Guide.- 20 Electropermeabilization and Electrosensitivity of Different Types of Mammalian Cells.- 21 Molecular Genetic Applications of Electroporation.- 22 Plant Gene Transfer Using Electrofusion and Electroporation.- 23 Electric Field-Induced Fusion and Cell Reconstitution with Preselected Single Protoplasts and Subprotoplasts of Higher Plants.- 24 Critical Evaluation of Electromediated Gene Transfer and Transient Expression in Plant Cells.- 25 Transformation Studies in Maize and Other Cereals.- 26 Cells in Electric Fields: Physical and Practical Electronic Aspects of Electro Cell Fusion and Electroporation.- 27 External Electric Field-Induced Transmembrane Potentials in Biological Systems: Features, Effects, and Optical Monitoring.

Electroporation and electrofusion in cell biology

Article de synthese sur le role des polyamines durant les phases de croissance et de developpement: division cellulaire embryogenese, rhizogenese, floraison et croissance du tube pollinique. Etude des relations entre polyamines et regulateurs de croissance des plantes

Do polyamines have roles in plant development

The considerable amount of activity in the field of electrofusion and electropermeabilization is very promising from the point of view of new insights into biomembranes and new technologies in the future for the production of new compounds and modification of cell systems for nutrition, energy production and the removal of waste products. It is particularly gratifying to see how basic science has provided the foundation for a useful technology, although in some cases the time needed to develop an application is very long. In other cases, it is necessary to overcome the difficulties posed by existing schools of thought which have been shown to be wrong. It is fascinating to observe the many developments and discoveries in the areas of physics, material science, space technology and electronics which are just waiting to be applied to biological systems. An increased interdisciplinary collaboration between physicists and biologists could provide considerable impetus to biology and its application in technology. However, this can only be achieved if basic research into biological membranes is accelerated. The techniques for electrical breakdown, electropermeabilization and electrofusion could be an important tool in this process, since we cannot rule out the possibility that the high electrical fields occurring naturally in the membrane play an important role in the selective transport of substances across the membrane as well as in natural regulatory processes.

Electrical breakdown, electropermeabilization and electrofusion

In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.

/pdf/stress-and-defense-responses-in-plant-secondary-metabolites-29akr1xmji.pdf

Stress and defense responses in plant secondary metabolites production

Abscisic acid (ABA) plays a significant role in the regulation of many physiological processes of plants. It is often used in tissue culture systems to promote somatic embryogenesis and enhance somatic embryo quality by increasing desiccation tolerance and preventing precocious germination. ABA is also employed to induce somatic embryos to enter a quiescent state in plant tissue culture systems and during synthetic seed research. Application of exogenous ABA improves in vitro conservation and the adaptive response of plant cell and tissues to various environmental stresses. ABA can act as anti-transpirant during the acclimatization of tissue culture-raised plantlets and reduces relative water loss of leaves during the ex vitro transfer of plantlets even when non-functional stomata are present. This review focuses on the possible roles of ABA in plant tissue culture and recent developments in this area.

The role of abscisic acid in plant tissue culture: a review of recent progress

Genotype, age of tree, nature of explant and size (length and diameter), season of explant collection, explant position on medium, plant growth regulators and certain additives (ascorbic and citric acids, adenine sulphate, L-arginine, glutamine and ammonium citrate), incubation conditions, and subculturing period greatly influenced the in vitro clonal propagation of P. cineraria. The maximum number of 10–12 shoots were induced from the nodal shoot segment from pruned thorny adult trees on Murashige and Skoog's (MS) medium containing 0.1 mgl-1 indole- 3-acetic acid (IAA)+2.5 mgl-1 benzylaminopurine (BAP)+additives. Higher temperature (31+-2°C) and mixed (fluorescent and incandescent) light of 50 μmol m-2 s-1 photon flux density for 12 h per day photoperiod favoured shoot induction and subsequent growth. Explants from thornless trees produced 6–8 shoots per explant on MS medium containing 0.1 mgl-1 IAA+5.0 mgl-1 BAP + additives. Nodal shoot segments obtained from root and stump sprouts produced multiple shoots. Root segments differentiated into multiple shoots on MS medium containing 0.5 mgl-1 indolebutyric acid (IBA)+2.5 mgl-1 BAP.

Factors affecting in vitro clonal propagation of Prosopis cineraria

A newly developed and novel DNA marker technique, i.e. start codon targeted (SCoT) polymorphic markers that target plant gene regions were used to assess genetic stability of in vitro raised plants of Cleome gynandra multiplied by enhanced bud proliferation from nodal segments. Seven randomly selected micropropagated plants, following at least 2 months of growth in the greenhouse along with mother plant were subjected to molecular analysis. Of 24 primers screened, 15 primers produced unambiguous and reproducible bands. All 15 primers generated a total of 65 fragments, with a mean of 4.3 ranging 2–7 per primer. No polymorphism was detected in regenerated plants and the mother plant, revealing the genetic fidelity of the in vitro raised plantlets. To verify the results of SCoT analysis, random amplified polymorphic DNA (RAPD) markers were also used for the assessment of genetic fidelity of tissue culture raised plants. The monomorphic banding pattern in micropropagated plants and the mother plant obtained from SCoT and RAPD analysis confirms the genetic stability of the in vitro raised plants and demonstrates the reliability of our micropropagation system for C. gynandra, an important C4 plant.

Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis

To evaluate genetic homogeneity of 1-year-old guava (Psidium guajava L.) plants developed from in vitro somatic embryogenesis, DNA from leaf tissues of seven randomly selected plants along with the mother plant, was isolated and subjected to molecular analysis. A total of six Simple Sequence Repeat (SSR) primer pairs, producing reproducible and clear bands ranging from 100 to 300 bp in size, resulted in amplification of single band (allele), corresponding homozygous individuals. Moreover, of 10 different inter-simple sequence repeat (ISSR) primers screened, six produced resolvable, reproducible and scorable bands. All these ISSRs produced a total of 25 bands, ranging between 300 and 1,200 bp length, and the number of scorable bands, for each primer varied from three to six with an average of 4.1 bands per primer. The amplification products were monomorphic across all the micropropagated plants produced by all SSR and ISSR primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for guava.

Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers

A reliable and reproducible protocol for in vitro regeneration has been developed for Alhagi maurorum, a rare and medicinally important plant of family fabaceae. MS medium with BAP (2.0 mg l−1) proved to be the best for shoot bud induction from nodal segments. The rate of shoot multiplication was found to be influenced by a number of factors, viz., media composition, plant growth regulator’s type and concentration, successive transfer of mother explant for different passage, culture vessels and gelling agents. Modified MS medium (modified having nitrates reduced to half) solidified with 0.14 % gelrite and containing BAP (0.5 mg l−1), IAA (0.1 mg l−1) and additives was found optimum for shoot multiplication which gave rise to maximum number of shoots (33.5 ± 3.43 per culture vessel). The in vitro regenerated shoots were rooted under both in vitro (on half strength MS salts with 1.0 mg l−1 IBA + 100 mg l−1 activated charcoal) as well as ex vitro (on sterile soilrite by treating shoot base with 250 mg l−1 each of IBA and NOA for 4 min in green house) conditions. Thereafter, the in vitro and ex vitro rooted plantlets were hardened under green house conditions with 70 and 90 % survival rate, respectively. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity of seven tissue cultured plantlets growing in green house condition with mother plant. The amplification products were monomorphic across all the seven micropropagated plants as well as mother plant produced by all SCoT, ISSR and RAPD primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for A. maurorum. To the best of our knowledge, this is the first report on micropropagation and genetic homogeneity assessment of A. maurorum, which can be applied for large scale multiplication of elite genotypes of A. maurorum.

Narpat S. Shekhawat

Papers

The role of abscisic acid in plant tissue culture: a review of recent progress

Factors affecting in vitro clonal propagation of Prosopis cineraria

Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis

Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers

Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers