viewed as a primitive sexual process, since it involves interaction of homologous dna from two individuals to form recombinant dna that is passed on to succeeding generations. bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes (see evolution of sexual reproduction; meiosis.) methods and mechanisms of transformation in laboratory schematic of bacterial transformation – for which artificial competence must first be induced. bacterial artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to dna by exposing it to conditions that do not normally occur in nature. typically the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock). calcium chloride partially disrupts the cell membrane, which allows the recombinant dna to enter the host cell. cells that are able to take up the dna are called competent cells. it has been found that growth of gram-negative bacteria in 20 mm mg reduces the number of protein-to-lipopolysaccharide bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation. the role of lipopolysaccharides here are verified from the observation that shorter o-side chains are more effectively transformed – perhaps because of improved dna accessibility. the surface of bacteria such as e. coli is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the dna is also negatively charged. one function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a dna molecule to adhere to the cell surface. dna entry into e. coli cells is through channels known as zones of adhesion or bayer's junction, with a typical cell carrying as many as 400 such zones. their role was established when cobalamine (which also uses these channels) was found to competitively inhibit dna uptake. another type of channel implicated in dna uptake consists of poly (hb):poly p:ca. in this poly (hb) is envisioned to wrap around dna (itself a polyphosphate), and is carried in a shield formed by ca ions. it is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to dna. the heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the dna to enter the cells through either cell pores or the damaged cell wall. electroporation is another method of promoting competence. in this method the cells are briefly shocked with an electric field of 10-20 kv/cm, which is thought to create holes in the cell membrane through which the plasmid dna may enter. after the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms. yeast most species of yeast, including saccharomyces cerevisiae, may be transformed by exogenous dna in the environment. several methods have been developed to facilitate this transformation at high frequency in the lab. yeast cells may be treated with enzymes to degrade their cell walls, yielding spheroplasts. these cells are very fragile but take up foreign dna at a high rate. exposing intact yeast cells to alkali cations such as those of caesium or lithium allows the cells to take up plasmid dna. later protocols adapted this transformation method, using lithium acetate, polyethylene glycol, and single-stranded dna. in these protocols, the single-stranded dna preferentially binds to the yeast cell wall, preventing plasmid dna from doing so and leaving it available for transformation. electroporation: formation of transient holes in the cell membranes using electric shock; this allows dna to enter as described above for bacteria. enzymatic digestion or agitation with glass beads may also be used to transform yeast cells. efficiency – different yeast genera and species take up foreign dna with different efficiencies. also, most transformation protocols have been developed for baker's yeast, s. cerevisiae, and thus may not be optimal for other species. even within one species, different strains have different transformation efficiencies, sometimes different by three orders of magnitude. for instance, when s. cerevisiae strains were transformed with 10 ug of plasmid yep13, the strain dkd-5d-h yielded between 550 and 3115 colonies while strain os1 yielded fewer than five colonies. plants a number of methods are available to transfer dna into plant cells. some vector-mediated methods are: agrobacterium-mediated transformation is the easiest and most simple plant transformation. plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for ten minutes in a fluid containing suspended agrobacterium. the bacteria will attach to many of the plant cells exposed by the cut. the plant cells secrete wound-related phenolic compounds which in turn act to upregulate the virulence operon of the agrobacterium. the virulence operon includes many genes that encode for proteins that are part of a type iv secretion system that exports from the bacterium proteins and dna (delineated by specific recognition motifs called border sequences and excised as a single strand from the virulence plasmid) into the plant cell through a structure called a pilus. the transferred dna (called t-dna) is piloted to the plant cell nucleus by nuclear localization signals present in the agrobacterium protein vird2, which is covalently attached to the end of the t-dna at the right border (rb). exactly how the t-dna is integrated into the host plant genomic dna is an active area of plant biology research. assuming that a selection marker (such as an antibiotic resistance gene) was included in the t-dna, the transformed plant tissue can be cultured on selective media to produce shoots. the shoots are then transferred to a different medium to promote root formation. once roots begin to grow from the transgenic shoot, the plants can be transferred to soil to complete a normal life cycle (make seeds). the seeds from this first plant (called the t1, for first transgenic generation) can be planted on a selective (containing an antibiotic), or if an herbicide resistance gene was used, could alternatively be planted in soil, then later treated with herbicide to kill wildtype segregants. some plants species, such as arabidopsis thaliana can be transformed by dipping the flowers or whole plant, into a suspension of agrobacterium tumefaciens, typically strain c58 (c=cherry, 58=1958, the year in which this particular strain of a. tumefaciens was isolated from a cherry tree in an orchard at cornell university in ithaca, new york). though many plants remain recalcitrant to transformation by this method, research is ongoing that continues to add to the list the species that have been successfully modified in this manner. viral transformation (transduction): package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. if the genetic material is dna, it can recombine with the chromosomes to produce transformant cells. however, genomes of most plant viruses consist of single stranded rna which replicates in the cytoplasm of infected cell. for such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. the progeny of the infected plants is virus-free and also free of the inserted gene. some vector-less methods include: gene gun: also referred to as particle bombardment, microprojectile bombardment, or biolistics. particles of gold or tungsten are coated with dna and then shot into young plant cells or plant embryos. some genetic material will stay in the cells and transform them. this method also allows transformation of plant plastids. the transformation efficiency is lower than in agrobacterium-mediated transformation, but most plants can be transformed with this method. electroporation: formation of transient holes in cell membranes using electric pulses of high field strength; this allows dna to enter as described above for bacteria. fungi there are some methods to produce transgenic fungi most of them being analogous to those used for plants. however, fungi have to be treated differently due to some of their microscopic and biochemical traits: a major issue is the dikaryotic state that parts of some fungi are in; dikaryotic cells contain two haploid nuclei, one of each parent fungus. if only one of these gets transformed, which is the rule, the percentage of transformed nuclei decreases after each sporulation. fungal cell walls are quite thick hindering dna uptake so (partial) removal is often required; complete degradation, which is sometimes necessary, yields protoplasts. mycelial fungi consist of filamentous hyphae, which are, if at all, separated by internal cell walls interrupted by pores big enough to enable nutrients and organelles, sometimes even nuclei, to travel through each hypha. as a result, individual cells usually cannot be separated. this is problematic as neighbouring transformed cells may render untransformed ones immune to selection treatments, e.g. by delivering nutrients or proteins for antibiotic resistance. additionally, growth (and thereby mitosis) of these fungi exclusively occurs at the tip of their hyphae which can also deliver issues. as stated earlier, an array of methods used for plant transformation do also work in fungi: agrobacterium is not only capable of infecting plants but also fungi, however, unlike plants, fungi do not secrete the phenolic compounds necessary to trigger agrobacterium so that they have to be added, e.g. in the form of acetosyringone. thanks to development of an expression system for small rnas in fungi the introduction of a crispr/cas9-system in fungal cells became possible. in 2016 the usda declared that it will not regulate a white button mushroom strain edited with crispr/cas9 to prevent fruit body browning causing a broad discussion about placing crispr/cas9-edited crops on the market. physical methods like electroporation, biolistics ("gene gun"), sonoporation that uses cavitation of gas bubbles produced by ultrasound to penetrate the cell membrane, etc. are also applicable to fungi. animals introduction of dna into animal cells is usually called transfection, and is discussed in the corresponding article. practical aspects of transformation in molecular biology the discovery of artificially induced competence in bacteria allow bacteria such as escherichia coli to be used as a convenient host for the manipulation of dna as well as expressing proteins. typically plasmids are used for transformation in e. coli. in order to be stably maintained in the cell, a plasmid dna molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the replication of the cell's own chromosome. the efficiency with which a competent culture can take up exogenous dna and express its genes is known as transformation efficiency and is measured in colony forming unit (cfu) per μg dna used. a transformation efficiency of 1×108 cfu/μg for a small plasmid like puc19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed. in calcium chloride transformation, the cells are prepared by chilling cells in the presence of ca2+ (in cacl2 solution), making the cell become permeable to plasmid dna. the cells are incubated on ice with the dna, and then briefly heat-shocked (e.g., at 42 °c for 30–120 seconds). this method works very well for circular plasmid dna. non-commercial preparations should normally give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/μg or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of plasmid. protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 109. the chemical method, however, usually does not work well for linear dna, such as fragments of chromosomal dna, probably because the cell's native exonuclease enzymes rapidly degrade linear dna. in contrast, cells that are naturally competent are usually transformed more efficiently with linear dna than with plasmid dna. the transformation efficiency using the cacl2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid dna. cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the electroporation process. selection and screening in plasmid transformation because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. the plasmid therefore requires a selectable