Warning: The following article has been machine-translated from Italian, and the English version has been only partially checked.
The genetic manipulation of plants has been practised for hundreds of years. Agronoms and horticulturalists have developed schemes of hybridisation among plants to introduce and to maintain some desired characteristics. The classical methods are slow and uncertain, they require sexual reproduction followed by repeated recrossings between progeny and progenitors and they also sometimes transfer unwanted characteristics. Scientific developments have allowed new techniques, mostly used today for producing many plants from one with particular characteristics. Obtaining just these characteristics, together with speeding the propagation of the plants, is the purpose of modern biotechnology.
Among the most obvious characteristics looked for:
- Increased quality and yield of the crop
- Increased tolerance of environmental pressures (salinity, extrem temperature, drought)
- Resistance to viruses, fungi and bacteria
- Increase tolerance of insects
- Increased tolerance of herbicides
Asexual reproduction determines the aggressiveness of a species in colonizing the nearby environment, with evolutionary advantages because the offspring are all clones of the parent. In a lot of multicellular cryptogams (fungi, lichens, bryophytes and pteridophytes) this happens through spores, produced following mitosis and contained in the sporangia, in the [cormofites] (bryophyta, pteridophyta and spermatophyta) through parts of the body of the plant that once fallen to earth are able to take root.
The scion is a portion of a plant (usually a stem or branch) cut immediately under a knot, without leaves, and buried. The result of the operation depends on persuading it to take root. Layering consists of making the buried part of the plant take root while it is still united to the parent plant. The buried branch or stem is surrounded by a mound of earth or well dampened peat in which the new roots will grow. This is therefore a more rapid and efficient method than the scion.
Evidently these methods do not bring genetic changes, but serve only to clone the plant.
The use of the grounds of crop with the vegetable cells has allowed the researchers to study the so-called Calluses, de-differentiated cellular heaps propagated in vitro to the endless one. to get the calluses first of all needs to explant cells from the plant. According to the kind he/she is preferred to choose among:
Through the passage to protoplasm, the cells fabric-specifications of the plant differentiation in totipotent cells, able that is to express any part of their genetic information. This characteristic, together with the ability to form crowd you cellular immortal, it differentiates the vegetable cells from those animal. Every cell of a callus can regenerate the whole one cried from which it has been isolated in two principal ways:
1. somatic embriogenesis: from the callus it originates an embryo, that develops him then in adult plant (for example the carrot);
2. somatic organogenesis: from the callus an organ regenerates him, usually leaflets, and subsequently the rest of the plant (for example tobacco).
In both cases the growth in vitro happens in the FITOTRONs, sterile climatic cells where temperature, damp and light-dark cycle are checked and programmable. With the somatic embryogenesis the vegetable embryos of certain kinds can be encapsulated (as with carrots, celery and tomatoes) to get artificial seeds. An important part is up to the vegetable hormones, whose role has been open really thanks to the analysis of the behavior of the cells in crop; the production of roots is checked by the auxine class of hormones, while the CYTOKININEs regulate the growth of the buds and therefore of the stem. Them as inductors can be used in the scions and layerings.
And evident therefore that these techniques of micropropagation allow to produce a lot of plants, also million, in the turn of few months. Besides the possibility is had to avoid that infected plants are simply formed using meristematic fabrics or withdrawing fabrics in the healthy zones of the plant and cultivating in presence of antibiotics and antivirals.
Isolating the hybrids so gotten you/he/she can be propagated in crop a Cellular Line it Interbreeds, as more stable as more similar they were the organisms of departure. Subsequently to the fusion of the nucleuses, during the following cellular divisions, the geniuses of one of the two kinds are progressively eliminated.
This has allowed the study of the genetic expression in different cellular environments of the usual one and above all of the organization of the [genoma]: checking what chromosome or fragment of chromosome has been lost, it is possible to word a genetic map of the chromosome that contains her/it location of every single gene.
Despite this obstruction to the formation of a completely hybrid plant, you/he/she is seen that a fraction of the geniuses can be preserved with formation of the so-called Asymmetrical Hybrid. You gets so a plant with all the characteristics of a progenitor anymore some line of the other. In this way it is possible to transfer useful quality even if the sexual hybridization doesn't allow him/it.
The hybridization sexual other is not but the pollination driven by the man of a plant of variety or, when it is possible, of different kind. They gathers so the positive quality of different variety, selecting then for different generations up to stability [genotipica] and [fenotipica].
Damaged cells of a plant (for [es]. from a lesion) they free factors that activate in the bacterium the geniuses [vir], of the virulence, located in the [PLASMIDE] Ti, [tumor inducing]. Proteins will be synthesized with function of factors of transcript.
Plasmids are molecules of circular DNA [extracromosomico] not tightly necessary to the bacterias, but that they bring advantages. They confer antibiotic resistance, [patogenicità], ability to metabolize in nourishing unusual substances, etc... they are transmissible is vertically from a bacterium to his/her/their progeny, that horizontally from bacterium bacterium. In genetic engineering as vectors are used for the transport of DNA.
This plasmid also contains a segment denominated T-DNA, carrying genes for the synthesis of [OPINE], of enzymes that degrade the [opine] and of [fitormoni], among which the [auxina].
Segment of the [plasmide] Ti
The T-DNA following the production of the [TFs] (factors of transcript) of the virulence, [escisso] comes awry to the height of the two Sequences [BORDER] and to valley of the same T-DNA, that passes as single filament in the vegetable cell in a trial similar to the bacterial conjugation. The [plasmide] is sheltered for [replicazione] of the DNA. Once inside the cell, the T-DNA has to enter the nucleus and to integrate himself/herself/themselves with the cellular DNA in a casual site, usually in multiple copies. This overturns the metabolism of the cell putting her/it to the service of his sophisticated parasitic genetic. The induced vegetable hormones are the cause of the growth not regulated.
Recapitulating, the process of infection is composed of:
Integrative plasmid
Binary vector
The buds develop him in few weeks, they transfer him therefore in terrestrial that induces the formation of the roots ([auxine]). The whole trial takes from 4 to 7 weeks and is applicable to an ample variety of [dicotiledoni].
Clonal Propagation
Asexual reproduction
Among the most ancient and widespread methods of plant propagation we find scions and layerings, which are really shoots produced by asexual (agamic) reproduction of the [cormofites]. Micropropagation
The original plant in this trial is not destroyed. Fundamental it is to sterilize the withdrawn fabrics, to avoid that the rapids growth of the present bacterias on the surface of the plants suffocates the growth of the callus; mincing and treating with [cellulasi] the protoplasto frees him, that begins to reconstruct him the wall after a few times (obviously if the enzyme is removed by the medium). Made the new wall the cell it begins to divide itself if the correct nutrient substances are present (salts, sugars and vitamins) and traces of vegetable hormones. They will begin so to form him some buds, that can be still separated and essayed with cellulase for a new cycle of propagation. Calluses can be gotten both from somatic cells and from sexual cells. somaclonal variability
Often the plants gotten for micropropagation are not genetically identical to the original one. They occur with greater frequency mutations when the cells are cultivated in vitro. This somaclonal variability is considered profit, in how much a tool is for getting best plants of the original one. you distinguishes:
Obviously not all the mutations will be advantageous, but among the many that happen someone it will be him/it, and you/he/she can be selected her/it. As it is unlikely that the same mutation happens on both the alleles of a diploid plant (not to mention tetraploid plants ...), when he/she is wanted to exploit the phenomenon of the somaclonal variability to get best mutants they are cultivated in vitro aploid cells as the immature pollen. Dosing properly the vegetable hormones is induced the embryogenesis of aploid plants. This facilitates the recognition of mutations, considering that the heterozygote can disguise its phenototype. Once [riconsciuta] and isolated the changed plant the state diploid you/he/she can be reestablished with the [COLCHICINA], an alkaloid derived by the plant of the autumn crocus that ties it to the molecules of [tubulina] preventing the formation of the fused mitotic and therefore the [citodieresi]. The [cromatidis] brothers separate him and they become homologous chromosome. This technique is note with the name of [androgenesi]. A lot of kinds of commercial interest (more than a hundred) they bring improvements gotten in this way, among which you/they detach tobacco, bowline, life, potato, laughed, maize and wheat. Mechanisms that produce variability
Changes in the [ploidia] of the cells in crop
Owed to:
A high rate of these phenomena doesn't correspond to a high percentage of cells or so-called polyploids. This is due to diploid selection: in a mixed population of cells with different ploidies, the diploids preserve better their potential [organogenetico] than the polyploids, probably because of an increased ability to form meristems. artificial rearrangement of tissue layers
A lot of horticultural plants are chimeras [PERICLINALI], that is they have suffered a mutation in a meristem cell and this has given origin to a different layer from the preceding and from the following one. The cells of the meristems in fact can divide anticlinally (perpendicularly to the surface) or periclinally (in parallel); if a continuous cell to divide itself periclinally gives origin to a layer of fabric. These layers can mix during a rapid proliferation as that of the calluses (if you/he/she is used really that meristems). Therefore regenerated plants with these unforeseen event can contain a different artificial composition or even not to be more artificial. structural changes in the sequence of the DNA
They are induced from radiations and from chemical substances but you/they can be also spontaneous. The coarse alterations of the genome are the principal cause of the variability [somaclonale]. We distinguish:
These alterations as the changes of ploidy, increase to increase some duration of the crop.
phenotypic epigenetic alterations
They are temporary and reversing changes, but you/they can persist for the whole life of the regenerated seedling. Common is the phenomenon of the [RINGIOVIMENTO], especially in woody kind (gymnosperms), that brings to morphological differences, precocious flowering, increase of the formation of temporary roots and the vigor of the plant. The causes of these non-hereditable alterations are not known, but probably induced by the environment of crop. In vitro selection
With this method a plant resistant to illnesses, insects, and environmental stress can be gotten. It implicates the to submit a population [calliforme] to a proper selective pressure (as the growth in small doses of herbicides) and the recovery of a varying line of cells that has developed resistance or tolerance to the stress. You benefits some speed of propagation in crop of the calluses to effect a real natural selection (spontaneous mutations) or to get variability with chemical or physical agents (induced mutations). The fields of search try to select resistant lines to the salinity, to the cold, to the herbicides, to the heavy metals,... cellular hybrids
The contained genetic information in cells of different origin can be combined in a single nucleus through cellular fusion. Cellular fusion requires that some cells come into contact and it includes a brief destruction of the cellular membranes using chemical agents. When the reconstitution of the membranes happens, the adjacent cells can reform together their membranes producing a single hybrid cell. Initially the cell derived by fusion will contain two nucleuses ([ETEROCARIONTE BINUCLEATO]), but after the division [cellulare1.3] the chromosomal outfits of the two cells come him to find inside a single nucleus ([SINCARIONTE]). And' possible to perform the fusion both on two types of cells that belong to the same kind (he speaks to this case of inter-species hybrid) that on two types of belonging cells to different kind. In the first case the hybrid cell will preserve the whole chromosomal order of the two cells of departure, while in the second case the cell fused curtains to eliminate the belonging chromosome to a type of cell. The products of the fusion more of two cells generally has scarce possibilities of survival. You/they can be interbred any cell with any other, without limits; have been made very fanciful experiments, in which cells of man-mouse were gotten, cells animal-vegetali1.4 and even with microorganisms! Recombinat DNA
[Agrobacterium]
[Tumefaciens]
Him to. [tumefaciens] is a Gram-negative aerobic bacterium that induces tumors in the zone of infection, known with the name of Gall Of the Collar, that is of the part of border between stem and roots. The infected cells acquire the ownership to grow in way not regulated, and they maintain her/it even if the bacterium is not more present; I am therefore real cells [tumorali] whose [genoma] has been however integrated with extraneous portions. -----------------------------------------------------------
|Genes |Left | T-DNA |Right |
| | |--------------------------| |
|Vir |Border | Opine | Enzymes | Ormons |Border |
-----------------------------------------------------------
The synthesis of [opine] happens thanks to some enzymes codified by the T-DNA which you/they bring to the modification of certain amino acids that only the bacterium [infettante] is able to metabolize as source of carbon and nitrogen. [Rhizogenes]
A. [Rhizogenes] exists, also another [Gram] negative that contains to the place of the [plasmide] You the [PLASMIDE] RI, [root inducing]. With the same procedure it drives the cells in which it is integrated to differentiate him in roots, said [HAIRY ROOTS], hairy roots, also in which [opine] are produced. From roots [hairy root] induced by the bacterium has been possible to get, directly or more often by crop of fabrics, whole plants containing [transgeniche] the T-DNA [trasformante]. The analysis [fenotipica] of these individuals has put in evidence a series of modifications characteristic said Phenotype [HAIRY ROOT]. Even though with some differences among the studied kinds, the plants [transgeniches] introduce a more intense green color in the leaves, a smaller height for shortening of the [internodis], loss of the dominance [apicale] with greater development of the gems [ascellari], leaf shriveling, one accented[rizogenesi] with partial loss of the radical [geotropismo] 2.1, a reduction of the fertility and the production of seed, and in kind [bi] - and [poliennali] a reduction of the period of life. All of this would not do certain to think to a possible use of a group of geniuses able to instigate so negative alterations of the plant. The T-DNA of A. [rhizogenes], of which the sequence is known [nucleotidica], is composed of 18 [OPEN READING FRAMES] ([ORFs]), that is presumably of 18 different functions [geniche], the dissection of which and their transfer in plant has allowed to ascertain that the phenotype [hairy root] can be induced moving only to the plant three of the 18 present geniuses in the T-DNA, particularly [ORF] 10, 11, 12 (according to a genetic definition [rol] Á., [B], [C], where [rol] is for [root locus]) and that the intensity of the modifications is function both some number of copies of the fragment of T-DNA foresees is of the level of expression of the geniuses themselves. method of the [cointegrazione]
Seen the premises him it is therefore well thought to exploit these knowledges to introduce an useful character in a plant. This method has been developed for avoiding the in partnership problems to the manipulation of fragments of DNA as great as the [plasmide] You. The T-DNA has been cloned in a standard vector of E. coli together with the gene [NPT2] (for the resistance to the [kanamicina]), to the gene [pBR322] (for the resistance to the [ampicillina]) and to the gene of interest. The result is an integrative said [plasmide].
Operations are simple of you ransom-sew you effect with him enzymes of restriction you adapt, that is that they separate the I fragment smaller possible without touching the useful gene. --------------------------------
| | | | |
--------------| T-DNA | gene | NPT2 | pBR322 |--------------
| | | | | | |
| -------------------------------- |
| |
------------------------------------------------------------You transforms the [plasmide] (what in test-tube is) in E. coli and they are selected the bacterias transformed with the [ampicillina] (and here it comes us profit the marker that gives the resistance to the [ampicillina], [pBR322]). they put on these to contact with intact [Agrobacteria], and, under proper conditions for the conjugation, the recombinant [plasmide] moves to [Agrobacterium], that will now have his You normal and the integrative [plasmide]. Both have the fragment T-DNA, whose sequences can interact for giving homologous recombination, that is the fusion of the integrative [plasmide], great around 5 [kilobasis] ([kb]), with the biggest [plasmide] You (200 [kbs]). The [plasmidis] that don't integrate him don't accumulate because they miss of an origin of [replicazione] for [Agrobacterium] (the so-called [oriC] in E Coli). they select you with the [kanamicina] the containing [Agrobacterias] the recombines [plasmide] You (and here it returns us profit the other used marker, [NPT2]). The system is extraordinarily [efficente], up to the 50% of the treated [protoplastis] it contains and it expresses the DNA transferred by the [Agrobacterium]. this tall efficiency of transformation allows us to select and to easily clone the modified [protoplastis].Binary system
And today the standard method for the transfer of the T-DNA. It makes use of two [plasmidis], Binary Vector and [PLASMIDE HELPER]. The binary vector is simply a [plasmide] You without the T-DNA, to the place of which they are inserted among the [borders] right and I damage the gene to move to the plant and a marker of selection. Another marker is inserted to the outside of the [borderses] for the future selection in E Coli. to notice that this vector maintains the origin of [replicazione] for [Agrobacterium]. -------------------------------------------------- ---------------
| Marcatore | left | gene | Marcatore | right | |Origine di |
---| batterico | border | | per calli | border |---|replicazione |---
| | | | | | | | | |
| -------------------------------------------------- --------------- |
| |
--------------------------------------------------------------------------
The [plasmide helper] is a [plasmide] You without the T-DNA but with still the geniuses [vir]. The binary vector is turned into E. coli, after selection the [trasformantis] are made to conjugate with a log of containing [Agrobacterium] the [plasmide helper] but not the You. In this way, following the activation from a wounded plant, the proteins of the geniuses [vir] (of the [plasmide helper]) they move the fragment of DNA among the two [borderses] (of the binary vector) in the vegetable cell. The binary vector, that is the containing [plasmide] the DNA to be transferred, as vector is maintained that is separately replied in [Agrobacterium]; in this difference is with the method of the [cointegrazione]. technique of the leaf disks
It is not easy to make to grow whole plants beginning from [protoplasti], also for the most proper kinds. An improvement was had him/it with this technique, considering that the leaves are a good source of cells [rigeneranti]. They cuts out small disk forms from the leaves, whose borders are quickly infected if it is inoculated with [Agrobacteria]. The disks are transferred then on filter paper it posts above cells nurses that produce factors of growth. After 2-3 days of crop it transfers him in terrestrial [stimolatore] of buds ([citochinine]) where the cells that bring the [plasmide] are selected thanks to a marker (antibiotic). not to take the risk to spread recombinant [Agrobacteria] in the environment is added to the crop an antibiotic as the [CEFOTAXIME] that kills the bacterium. markers of selection
The Geniuses Reporters furnish us support in to visualize the transformed cells. The gene of E. coli for the enzyme [ß-glucuronidasi] ([GUS]) you/he/she is often placed side by side to the DNA by [trasfettare] to the plants because they has invaluable levels of this enzyme. When cells that express the [GUS] are incubated with [X-glucuronide], a blue coloration [individuabile] produces him with methods [istochimici]. Or if a different substratum is used, the [GUS] can quantitatively be measured with a [fluorimetro]. Only disadvantage is that the cells must have killed for the analysis [istochimica]. Otherwise, using the gene of the [luciferasi] as gene reporter, to the addition of [ATP] and [luciferina] in the ground of crop light is produced, [rilevabile] also through a photographic film. Often, when he is already certain of a method and there is no need to confirm happens him transformation, they are used antibiotic so that to directly eliminate the cells not transformed. use of virus
The viruses would be the ideal solution to transfer of the DNA to all the cells of an adult plant, considering that they are suited in the evolution for to do really this. Unfortunately her almost totalities of the vegetable viruses are to [RNA]. Only two classes of virus containing DNA they are known, the virus of the Mosaic Of the Cauliflower ([caulimovirus]) and the [GEMINIVIRUSs]. The [caulimovirus] has a circular molecule of DNA of small dimensions, it spreads in the plant through the vascular system and you/he/she cannot be transmitted through the seeds. The last characteristic is very interesting because it allows to check the diffusion of the new geniuses, but the virus of the mosaic of the cauliflower has two great disadvantages, infected only some plant of the family of the cauliflower and it is able to transport in its [capside] alone brief sequences of DNA (300-400 bases). The [geminiviruses] have [genomi] served as two molecules to single helix of DNA, every of which it passes through a form [replicativa] to double helix. The molecule alone Á. is able of to reply him in the cells of the plant, but for the [infettività] it is in demand the molecule B. Poiché the DNA [replicativo] to double helix (molecule Á.) it is also infectious in absence of the protein covering, a lot of the regions that codify proteins of covering are able [venir] eliminated for making place to a [transgene]. The DNA Á can be then inserted among the sequences [border] of the T-DNA, and so the DNA [B] among other [borders], to constitute the binary vector, that will be used in the binary system. other
direct transformation
The fate of an introduced nucleic acid as such in a cell it is that to enzymatically be degraded quickly. Some cell, said competent cell, is under particular conditions and accepts the integration of the extraneous DNA in the [genoma]. The principal advantage is that it asks for little manipulation of the DNA, but it has a [fraquenza] of transformation to everything today rather low, around him 1%. [Microiniezione]
Maneuvering a thin needle of glass with mechanisms that allow moves [minimali] and constantly working to the microscope is possible to pierce the membrane of a cell without killing her/it and to inject you small quantities of DNA. The [microiniezione] can climb over the tied up problems to the use of the [protoplastis] and the crops in [vitro] with the important [monocotiledonis] for the agriculture. In theory it would be enough to inject the gene in the pollen and to install this in the ovary of the kind to get the seed [transgenico] and therefore the plant.
