Comparison of Gene Transfer Systems,Recombinant DNA Technology,Biolistics,Germline,Transgenic Plants,Southern Analysis,Molecular Analysis,Somatic Cells

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Comparison of Gene Transfer Systems

	Comparison of Gene Transfer Systems - Recombinant DNA technology is most advanced in tomatoes, potatoes, tobacco and rapeseed. Successful regeneration from fused cells has been achieved for maize, millet, rice and tropical forest legumes. A comparative account of various approaches for gene transfer. Thus far no transgenic cereals exist except for those recovered from protoplasts and direct gene transfer. Reliable data on configuration of transgenic plants obtained through the use of other gene transfer systems has not yet been published. What constitutes suitable proof? Neither genetic, nor phenotypic, nor physical data alone is acceptable as suitable proof of obtainment of transgenic plants. Proof of integrative transformation requires:
(1) serious controls for treatments and analysis; (2) tight correlation between treatment and predicted results; (3) tight correlation between physical (Southern blot, in situ hybridization) and phenotypic (enzyme assays) data; (4) complete Southern analysis containing (a) the predicted signals in high molecular weight DNA, in hybrid fragments between host DNA and foreign gene; and the complete gene and (b) evidence of the absence of contaminating DNA fragments or the identification of such fragments;
(5) data that allows discrimination between false positives and correct transformants in the evaluation of phenotypic evidence; and (6) correlation of the physical and phenotypic evidence with transmission to sexual offspring, as well as genetic and molecular analysis of offspring populations. Considerations of the biology of gene transfer may be helpful in understanding why some techniques work and others don't. It may also help in assessing the future potential of the various approaches. A transgenic plant can only result from integrative transformation in a totipotent cell or a cell that has a clonal connection to the "germline". Some 16 intrinsic problems have been enlisted by Protykus (1990) for obtaining integrative gene transfer in higher plants: (1) not all plant cells are totipotent; (2) plant cells differ in capacity to respond to triggers, a phenomenon termed competence; (3) cells from which it is hoped to regenerate transgenic plants must be competent forboth regeneration (in a broad sense) and integrative transformation;
(4) plant tissues are composed of cells competent for many different responses. Considering the two states of competence essential for recovery of transgenic plants the following situation has to be considered: (a) a very small minority of cells in plant tissues will be competent for both transformation and regeneration; (b) others will be competent for transformation or regeneration; (c) a larger fraction of the cell population will be potentially competent, which means that given the correct treatment they will have the potential to shift to the competent state; (d) a variable proportion of cells will not even be potentially competent but instead, noncompetent;
	(5) the relative composition of cell populations in tissues is determined by the genotype, type of organ, developmental state of the organ, and even the individual history of the experimental plant (6) the most effective trigger for shifting potentially competent cells to the competent state is mechanical (and enzymatic) wounding. Wound response is probably the biological basis for regeneration from somatic cells; (7) plant species differ in wound response as do different tissues of the same plant. Graminaceous plant species, especially cereals and maize, have only. a very rudimentary or no wound response; (8) for some genotypes it is possible to proliferate cells competent for regeneration under conditions that maintain this state. Such cell cultures contain cells competent for regeneration and (after protoplasting) competent for integrtive transformation; (9) plant cell walls are efficient barriers and traps of DNA molecules; (10) genes can be transported into cells across cell walls with the help of Agrobacterium, "biolistics", and microinjection; (11) production of transgenic plants requires efficient gene transfer into cells competent for regeneration and integrative transformation; (12) competence for integrative transformation is obviously very different from competence for transient expression; (13) nonviral DNA can integrate into the host genome. Its presence in a cell does not guarantee its integration; (14) nonviral DNA does not move from cell to cell but is restricted to the cell to which it has been delivered; (15) viral DNA (and RNA) moves from cell to cell.and can spread systematically throughout an entire plant. It is probably excluded from the meristems and the "germline" however; and (16) viral DNA does not integrate into the host genome even if present at a very high copy number.

System	Status	Remarks
Agrobacterium and dicots	A routine and efficient method for production of transgenic plants from numerous noncereal species.	Plants and tissues differ in wound response. Only plants and tissues with a pronounced wound response develop larger population of wound-adjacent competent cells for efficient transformation.
Agrobacterium and cereals	No transgenic cereals recovered so far	Tissues or population of cells which are either non-competent or potentially competent will not be converted into competent cells; wounding of differentiated cereal tissues does not lead to wound response induced differentiation in wound adjacent cells. Therefore no competent cells are available; instead, wounding leads to death of the wound adjacent cell
Agroinfection and dicots	Agroinfection can lead to transgenic plants via T-DNP integration.	Viral DNA integrated into T-DNA of Ti-plasmid of Agrobacterium can be delivered transfer process. T-DNA can integrate into plant cells with normal T-DNA and thus agroinfection can lead to integration of viral DNA in the wound-adjacent cell.
Agroinfection and cereals	This method has little potential for production of transgenic cereals.	Chances that agroinfection will produce transgenic cereals minimal; not different from normal Agrobacterium infection; if someone finds a way to induce integration of viral DNA or of foreign DNA integrated into replicating and spreading virus, somewhat hopeful.
Viral vectors	This method has little potential for production of transgenic cereals.	Viruses do not integrate into host genome and are excluded from meristems and thus from transmission to sexual offspring.
Incubation in DNA of seeds or embroys	Thus far, no transgenic plants have been recorded; not much potential.	Although experiments demonstrate the presence and expression of defined marker genes as well as replication of engineered viral DNA, they do not provide proof of integrative transformation.
Incubation in DNA of tissues or cells	No transgenic tissues or plants have been recovered; very low potential.	There have been many approaches whereby seedlings, organs, tissues, cells or cell cultures of numerous plant species have been brought into direct contact with foreign DNA and defined marker genes. The combination of several low frequency events will cause problems even if one step may occasionally work
Pollen tube pathway	No transgenic plants have been recovered; perhaps little potential.	Pollen tubes are not open pipes but sealed off with callose plugs; DNA trapped by cell wall material. There are probably nucleases not only in the synergids, but also in the pollen tube; there is no transport system known. However, the approach is attractive.
Liposome fusion with protoplasts and tissues	Transgenic plants have been with protoplasts and recovered from protoplasts but not from tissues and cells	DNA containing liposomes have been applied to various tissues, cell cultures and pollen tubes, with the rationale that liposome might help to transport via plasmodesma or directly across the cell wall. The approach is attractive.
Liposome infection	Thus far no transgenic tissue recovered.	This method has probably no advantage over straight forward microinjection, especially for production of transgenic cereals.
Protoplast and direct gene transfer	This method has only yielded transgenic cereals; still problematic because plant regeneration from protoplasts is difficult to achieve.	Competent protoplasts have been isolated from embryo genic suspension established from immature tissues. Standard direct gene transfer procedures with protoplasts from embryogenic suspension has led to regeneration of transgenic rice and maize. This is likely to be a problem for some years, however, because so far establishment of appropriate cell cultures is an art that also depends on parameters beyond experimental control.
Protoplasts from cereal plants	No transgenic controls have been recovered; no potential, to date.	DNA uptake is no problem as it can be shown easily with transient expression assays. If integration occurs, it has no consequences because protoplasts do not proliferate.
Microlaser	No transgenic tissue produced; not much potential.	As microinjection and biolistics definitely transfer DNA into walled plant cells, micro laser offers advantages in very specific cases in which those techniques are not applicable.
Electrophoresis into tissues	There is proof of integrative transformation; requires further experimentation.	Electrophoreses of DNA across the shoot meristem of barley seeds yielded indicative evidence in the form of radioactively labeled cell walls, positive GUS assays, and a protein of SPS-PAGE with E. coli GUS mobility.
Biolistics or particle gun	No transgenic offspring produced in cereals. This method has good potential for testing gene expression in transient systems.	Acceleration of heavy particles covered with DNA can be used to transport genes into plant cells and tissues. This technique caused some excitement because it was believed for some time that it would solve all gene transfer problems. Transgenic plants have been produced in soybean and tobacco and others will follow. This method is easy to handle; one shot can lead to multiple hits. The genes coated on particles resume biological activity. The target cells can be as different as pollen, cell culture, organ or meristem. Particles also reach deeper cell layers. Thus the method provides a biological vector-independent DNA delivery system into a great variety of cells.
Microinjection		Microinjection uses micro capillaries and microscopic devices to deliver DNA into defined cells in such a way that the injected cell survives and can proliferate. This technique has produced transgenic clones from protoplasts and chimeras from microspore derived pro embryos in oilseed rape. As with biolistics, microinjection delivers DNA into cells
Zygotic proembryos and Agrobacterium	Meristematic cells are not competent, integrative, transformation.	Transgenic tissue could not be detected either in the regenerated tobacco plant or in the sexual offspring.
Macroinjection	No proof for recovery of transgenic plants; probably no potential.	Very difficult to understand how the DNA could reach the sporogenic cells in the experimental design, as DNA would not only have to reach neighboring cells, but travel across many layers of cells.
Polar transformation	No transgenic plants have been produced; probably no potential.	Numerous experiments with defined marker genes have only given negative results.
Electroporation	No transgenic clones have been produced when applied to cells and tissues; not much potential with walled cells; routine method for gene transfer to protoplasts.	Protoplasts can be transformed with PEG, electroporation, microinjection and Agrobacterium. For protoplast systems electroporation is but one of several modifications of direct gene transfer. Since in numerous important cases plants can be regenerated from cell cultures and tissue expolants, but not from protoplasts, it has been important to test whether electroporation could transfer genes into walled cells. This does not appear to be the case.