Dierct Gene Transfers
2.3. Dierct Gene Transfers
Direct gene transfer may be defined as — ‘the introduction of DNA into plant cells without the involvement of any biological agent e.g., Agrobacterium, and leading ultimately to stable transformation’.
It has been duly observed that the spontaneous uptake of DNA by the plant cells is appreciably at a low ebb ; and, therefore, the much required both chemical and physical manipulations are afforded invariably so as to augment and facilitate the DNA to gain entry into the plant cells. Interestingly, the ‘gene con- struct’ that needs to be delivered right into the desired plant cells may be present or achieved in differ- ent manners, such as : (a) plasmid or cosmid vectors ; (b) bacteriophages ; (c) artificial yeast chromo- somes ; and (d) usage of native uncloned DNA for affecting strategic delivery.
Based on the survey of literature there are ten well-defined and distinct methodologies that are employed exclusively for the ‘direct transfer’, namely :
(i) Chemical methods ;
(ii) Electroporation ;
(iii) Particle gun delivery ;
(iv) Lipofection ;
(v) Microinjection ;
(vi) Macroinjection ;
(vii) Pollen transformation ; (viii) DNA Delivery via growing pollen tubes ; (ix) Laser-induced gene transfer ;
(x) Fiber-mediated gene transfer ; and (xi) Transformation by ultrasonication. Each of the aforesaid method shall be treated individually in the sections that follows :
PHARMACEUTICAL BIOTECHNOLOGY
2.3.1. Chemical Methods
It has been duly established that there are certain chemical substances, such as : polyethylene glycol (PEG), polyvinyl alcohol (PVA), and calcium phosphate [Ca 3 (PO 4 ) 2 ] predominantly increase the uptake of DNA by the plant protoplasts exclusively and squarely. PEG-Mediated DNA Delivery . The various sequential generalized steps adopted for the PGE- mediated DNA delivery are as stated under : (1) The plant protoplasts are suitably suspended in a transformation medium enriched with Mg 2+ ions instead of the Ca 2+ ions.
(2) Linearized plasmid DNA essentially having the ‘gene construct’ is carefully added into the protoplast suspension obtained in (1) above.
(3) To the resulting solution PEG (20% concentration) is added and pH adjusted to 8.0 (4) The ‘protoplasts’ thus obtained is subjected to a five-minute thermal-shock treatment at
45° C followed by an immediate transfer to ice just prior to the addition of DNA, because it significantly enhances the frequency of transformation by several orders of magnitude.
[Note : Carrier DNA incorporation at this specific stage does promote transformation ; however, in actual practice it is neither desirable nor necessary at all.]
(5) After a certain stipulated duration of ‘incubation’, the prevailing concentration of PEG is reduced adequately while that of CA 2+ ion is increased appreaciably in order to accomplish an enhanced transformation frequency.
Example : Transformation of tobacco protoplasts (Necotiana) : It has been observed that when the ‘synchronized tobacco protoplasts’ subjected to transfor-
mation either in the course of mitotic phase or S-phase give rise to the formation of 3% transformed colonies ; whereas, the corresponding ‘non synchronized tobacco protoplasts’ yielded only 1.5% effective transformation.
In actual practice, the treated protoplasts are ultimately cultured so as to regenerate adequately cell wall and thereby produce callus colonies from which the desired plants regenerated subsequently. It is, however, pertinent to state here that the proper use of a ‘selectable marker’ would certainly go a long way for the judicious selection of the transformed protoplasts in an appreciable extent.
2.3.2. Electroporation
Electroporation may be defined as — ‘the legitimate introduction of DNA into the cells by exposing them critically for specific very short durations directly to the electrical pulses of high-voltage field strength which perhaps induced transient pores in the plasma lemma ’.
Importantly, these generated/applied electrical pulses invariably enhance the permeability of protoplast membrane and evidently facilitates the logical entry of DNA molecules right into the cells, provided the DNA is in direct contact with the membrance desirably. In general, there are basically two marked and pronounced systems of electroporation, namely : (a) low-voltage long-pulses method ; and (b) high-voltage short-pulses method.
Example : Tobacco mesophyll protoplasts : The actual realistic values are as given below :
First instance :
Low-voltage long-pulses method :
300-400 V cm –1 for 10-15 ms (milliseconds ; exponential decay) ;
GENETIC RECOMBINATION
127 Second instance :
High-voltage short-pulses method :
1000-1500 V cm –1 for 10 µ s. (microseconds ; square-wave pulse generators) ;
Fig. 2.2(a) illustrates the details of the output characteristic featurs of the two principal types of electroporation methods in actual usage viz., high-voltage short pulse machine, and low voltage expo- nential decay machine ; wherein pulse duration (mS) is plotted along the X-axis and voltage (V) along the Y-axis.
Fig. 2.2(b) depicts the ‘circuit-diagram’ of a low voltage, exponential decay machine.
10 Pulse duration ( s) µ High voltage
lt a g e Vo
short pulse machine
Low voltage, exponential decay machine
Pulse duration (ms)
(a)
Power pack Capacitors
Electrodes Oscilloscope
(b)
Fig. 2.2(a). Output Characteristic Features of Two Major Type of Electriporation Devices ; and (b) Circuit Diagram of Low Voltage Decay Machine.
[Adopted from : D.Grierson, Plant Genetic Engineering, 1991]
Salient Features of Electroporation :
The various salient features of ‘electroporation’ are as enumerated under : (1) In a broader perspective, low-voltage long-pulses technique give rise to relatively high rates
of transient transformation ; whereas, high-voltage short-pulses method produce usually high rates of stable transformation.
(2) In several extensive and intensive studies in electroporation experimental parameters yield- ing almost 50% protoplast viable survival may invariably give the highest rates of stable
PHARMACEUTICAL BIOTECHNOLOGY
(3) The transformation frequencies may be critically enhanced by several-fold by means of the following two manners, namely :
(a) Sudden thermal-shock treatment (45°C) to the ensuing protoplasts just before electroporation, and
(b) Presence of low concentration (~ 8%) of PEG in the course of electroporation. (4) Certain plant species are found to be appreciably sensitive to PEG ; and hence, electroporation
may be adopted as the ‘method of choice’. (5) A plethora of plant species are amenable to PEG-induced gene transger (see section 2.3.1) ;
and, therefore, is regarded to be much more efficient, dependable and reliable in comparison to electroporation.
(6) Electroporation has been successfully and gainfully expoited to afford the production of fairly stable transformed cell-lines and/or plants in several plant species, such as ; maize, petunia, rice, tobacco, wheat, sorghum, etc. Example : For Tobacco : The frequency of transformation was quite high and ranged between 2-8% (in the presence of 7% PEG).
(7) Electroporation has been employed fruitfully to deliver strategically DNA into the intact plant cells.
(8) In accomplishing electroporation the corresponding transformation frequencies may be ad- equately improved upon by adopting any one of the following four means, namely : (a) employing field strength of 1.25 kV cm –1 ,
(b) incorporating PEG after addition of DNA, (c) thermal-shock treatment of protoplasts at 45°C for 5 minutes prior to the addition of DNA, and
(d) specifically employing ‘linear DNA’ instead of ‘circular DNA’.
2.3.3. Particle Gun Delivery [Synonyms : Biolistic or Ballistic Method of DNA Delivery]
In early 1990s, the particle gun delivery method was conceived and put into practice which essentially made use of a 1-2 µ m tungsten or gold particles, precoated with the DNA to be employed exclusively for transformation, are adequately accelerated to such a degree of velocities that gainfully enable their entry right into either the plant cells or the nuclei. In reality the actual ‘particle accelera- tion’ is accomplished by employing an appropriate and specific device that precisely varies both in design as well as function. There are two most effective and equally successful devices that usually aid in causing acceleration of the particles, namely :
(a) utilization of pressurized helium gas, and (b) utilization of the released electrostatic energy by a droplet of water after exposure to a very
high voltage. Interestingly, the relatively earlier devices actually employed blank cartridges via a sophisti-
cated and modified firing mechanism to afford the needed energy for the particle acceleration ; and this perhaps originated the nomenclature particle gun to this type of approach.
Helium pressurized device : The helium pressurized device mainly comprise of the following essential components, such as :
(iii) stopping screen ; (iv) macrocarrier carrying particles precoated with DNA ; and
(i) gas-acceleration tube ;
(ii) rupture disc ;
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Gas Acceleration Tube Rupture Disc
Macrocarrier Macroparticles [DNA-Coated]
Stopping Screen
Target Tissue
Fig. 2.3. A Helium Pressurized Device Particle Gun Involved in DNA Delivery Showing Macroparticles Coated with DNA being Conveyed in the Target Tissue.
The aforesaid components (i) through (v) are sequentially enclosed in a chamber so as to enable creation of partial vacuum that precisely facilitates particle acceleration and thereby causes reduction in damage to plant cells. Once the partial vacuum is affected in the gas acceleration tube, adequately pressurized helium gas is made to release in the said tube in order to break the strategically positioned rupture disc. In this manner the helium shock waves are generated that specifically accelerates the macroprojectile to which the DNA-coated microprojectiles are attached duly. Thus, the macroprojectile is adequately retained by a stopping screen, while the microprojectiles pass via this screen and ulti- mately get embedded right into the target tissue cells meticulously positioned 10 mm below the stop- ping screen.
Notes : (1) Helium is invariably preferable to air by virtue of the fact it is not only lighter but
also offers several advantages options. (2) In actual practice a 1000 psi (pounds per square inch) of pressurized helium gas is employed for causing acceleration.
Macrocarrier (Macroprojectile) : It is a 2.5 cm diameter having 0.06 mm thickness plastic membrane that is usually employed once only. Its light mass usually affords rapid advantageous acceleration.
Microparticles (Microprojectiles or Microcarriers) : They normally vary in diameter from 0.5 to 0.2 µ m. However, the average size of 1.0 µ m is employed commonly.
(a) Tungsten Particles : These are relatively cheaper, with usual irregular shape and size, found to be toxic to some specific cell types, and exhibit surface oxidation that may ultimately lead to precipitation of DNA. Besides, they have a tendency to form aggregates after the addition of DNA and this gives rise to the reduction of expected particle dispersion.
(b) Gold Particles : These are found to be comparatively more uniform in size (1-3 µ m) and shape. They usually exhibit lower toxicity, variable degree of coating with DNA, and hence are much more expensive.
DNA-Coating : The most critical factor with regard to the coating of microparticles with DNA is normally achieved through precipitation. In actual practice, it may be accomplished by mixing to- gether 1.25-18 mg microparticles and 0.5-70 µ g of the plasmid DNA in a calcium chloride (0.25-2.5 M) and spermidine (0.1 M) solution carefully. The resulting mixture is subjected to thorough mixing on a
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‘Vortex Mixer’ so as to ascertain uniform coating. After carrying out the DNA precipitation, the microparticles are meticulously transferred onto the surface of macrocarrier membranes, allowed to dry up and used almost immediately to obtain the best results,
Applications of Biolistic Technique : The various applications of the biolistic technique or the particle gun delivery are as follows :
(1) It helps to produce fairly stable gene transfers in a variety of vegetative species, namely ; cotton, maize, papaya, poplar, rice, soybean, sorghum, sugarcane, tobacco, wheat etc.
(2) Transfer of cry gene* from Bacillus thurigiensis into maize ; for resistance to European corn-borer, Ostrinia nubialis ; and that of bar gene** found in rice for causing resistance to phosphinothricin (herbicide).
(3) The particle gun gene transfer method is specifically found to be attractive and useful due to the fact that DNA can be strategically delivered right into the cells of shoot meristems located in short tips and embryos that particularly renders the gene transfer phenomenon absolutely independent of the prevailing regeneration ability of the species.
Note : (a) Biolistic technique is virtually applicable to all plants species and may even be
extended to the animal cells as well. (b) Its only major constrain/limitation being the rather expensive specialized accelera-
tion device (i.e., the particle gun).
2.3.4. Lipofection
Lipofection may be defined as — ‘the introduction of DNA into cells via liposomes’. It is consid- ered to be the ultimate method of choice for DNA delivery into the animal cells particularly when being cultured in vitro.
Lipofection also enjoys the reputation of being one of the initial few methods of delivering DNA into the plant protoplasts. However, the transformation frequencies of the order of 4 × 10 –5 have been observed. The integrated DNA evidently does not undergo rearrangements ; however, multiple copies
may obviously get integrated in tandem.*** Special Features : Following are the two special characteristic features of ‘lipofection’ namely : (1) Invariably, plasmid DNAs 9 kb,**** and of even larger dimensions have been adequately
integrated quite intact. (2) Higher transformation frequencies with PEG and electroporation render them even more
attractive.
2.3.5. Microinjection
In the event of microinjection, the DNA solution is injected almost directly right inside the cell by the help of capillary glass micropipettes using micromanipulators of a microinjection set of apparatus.
* Star Link corn is a transgenic product which contains the Bt gene Crg 9C derived from B. thurigiensis, that offers resistance against important corn insect pests e.g., European corn-borer.
** Phosphinthrocin acetyl transferase (PAT) coded by bar gene in Streptomyces spp. which detoxifies the herbicide L-phosphinothricin (PPT).
*** Arranged one behind another. **** Kilo base pairs.
GENETIC RECOMBINATION
It has been demonstrated amply via actual experimental procedures that it is much more convenient and efficacious to make use of ‘protoplasts’ instead of the ‘cells’ because the cell-wall specifically gives rise to serious interference with the process of microinjection.
Salient Features : Following are some of the most important salient features of microinjection, such as :
(1) Process of microinjection is technically not only demanding but also time-consuming i.e., one may microinject upto 50 protoplasts in a span of 60 minutes.
(2) Quite successful transformation by microinjection of cells and protoplasts has been accom- plished in Brassica sp., alfalfa, tobacco etc., with achievable transformation frequencies ranging from 14 to 66%. However, the corresponding results with cereals are rather not-so- encouraging.
(3) In order to achieve high transformation rates, it is a must to incorporate the DNA right into the nucleus or the cytoplasm of the specific cell. Hence, the success rate is found to be the highest with particularly the non-vacuolated embryonic cells and the densely cytoplasmic ones. A logical and plausible explanation with respect to the extremely low transformation rates amongst large vacuolated cells is due to the DNA being delivered into the vacuole directly which gets degraded consequently.
2.3.6. Macroinjection
Macroinjection may be defined as — ‘injection of plasmid DNA (or uncloned native DNA) right into the lumen of developing inflorescence* using a hypodermic syringe.’
It has been adequately advocated that the DNA is usually taken up by microspores in the course of certain particular state of their development. In 1987, an attempt was made to inject DNA specifically in the developing inflorescene of rye ; and eventually a rather much lower frequency (0.07%) of the transformed plants was recovered in the progeny. In reality, the concept of macroinjection is quite easy and simple ; however, the two glaring problems encountered are, namely : (a) poor attainable frequency ; and (b) inconsistency in achievable stable transformants.
Table : 2.1 summarizes the characteristic features with respect to the vital and pivotal DNA delivery techniques for the plant cells exclusively.
Table : 2.1. Comparison of Certain Vital and Pivotal DNA Delivery Techniques for the Plant Cells
DNA Delivery Techniques
S.No. Characteristic
Microinjection Particle Gun Features
Agrobacterium
Chemical and
Mediated
Electrical
1. DNA construct
— (Max. size)
< 50 kb**
5-20 kb
16 kb
2. Plant Range(s)
Restricted
All plants
All plants
All plants
3. Obtainable
— efficiency
Relatively high
Relatively high
* Complete flower head of a plant.
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4. Integrity of DNA Not affected by
High degree of insert
High degree of
5. Number of copies Invariably single High frequency
High frequency integrated
1 to 5
or a few copies
of multicopy
of multicopy
insertions.
insertions.
6. Special equipment
Yes required
NO
No (chemical)
Yes
Yes (Electrical)
7. Regeneration
Needed quite Not necessary protocol
(use of embryos and shoot tips)
May be required Not needed culture
8. Protoplasts
Not required
Necessary
9. Applicable to
Yes organized meristems
10. Applicability to
Applicability cereals
Applicable
Used frequently Possible
high
11. Chimeric plants
No
No
Quite often Quite often
(in case of
(in case meri-
meristems
stems bomarded)
employed)
[Adopted from : Biotechnology, Singh BD (1998)]
2.3.7. Pollen Transformation
It has been duly reported by some researchers that ‘gene transfer’ could be accomplished by simply allowing DNA to soak in pollen grains just prior to their actual usage for pollination. It is, however, pertinent to state here that these studies failed to be substantiated by other researchers who specifically made use of ‘cloned genes’. Though apparently the proposed methodology seems to be quite attractive bearing in mind its overall applicability and superb simplicity, yet till date there exists little definitive evidence and ample proof for a transgene being actually transferred by the air of pollen- soaked in the DNA solution.
2.3.8. DNA Delivery via Growing Pollen Tubes
In this specific technique, first of all the stigma* of a flower is carefully incised sometime after its pollination ; and secondly the DNA solution is duly smeared onto the incised surface painstakingly. In actual practice the accurate and precise ‘time of stigma excision’ shall virtually depend upon the rate of pollen-tube growth that may vary from 5-20 minutes to 2-3 hours.
* The spot on the ovarian surface where rupture of a graafian follicle takes place.
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Examples : (i) Rice : In this particular instance the plasmid DNA containing nptil gene was duly applied
onto the incised surface of the stigma. Consequently, 20% of the seeds thus obtained actually showed the presence of nptil gene in copy numbers varying from 1-300.
(ii) Barley : In this case, the following observations were made, namely :
_ transformation frequency ranged between 10 –4 to 10 of the seedlings thus accomplished _ expression level of nptil gene was rather low
_ both mature plants and their corresponding progeny failed to show any nptil expression.
Special Notes : Following are some of the ‘special notes’ with respct to the DNA delivery via growing pollen tubes :
(1) Melthodology may be promising, easy and simple but its integrity with respect to its
consistency in results and stable transformations are yet questionable, and (2) Two vital aspects, such as : mechanism of DNA transfer into the zygotes, and support-
ing factors affecting it must be studied more intensively and aggressively.
2.3.9. Laser-Induced Gene Transfer
The application of laser* has been employed effectively and successfully for high frequency (10 –3 ) transfection** of animal cells. In reality, the lasers puncture transient holes in the cell membrane spe- cifically via which DNA would gain entry into the cell cytoplasm. In fact, the usage of lasers to afford delivery of DNA into the plant cells was a pretty long practice ; however, there is little available information(s) with regard to its transient expression or stable integration.
2.3.10. Fibre-Mediated Gene Transfer
In this specific and articulated approach the DNA is strategically delivered into the cell cyto- plasm and nucleus by means of the silicon-carbide fibres having 0.6 µ m diameter and 10 µ m length.
The methodology essentially involves the intermixing of suspension culture cells and plasmid DNA having gus gene along with the silicon-carbide fibres, all of which are adequately suspended in the culture medium with utmost precautions. The ultimate mixture was thoroughly mixed in a vortex mixer, and the resulting cells were quantitatively estimated for their transient gus gene expression. The fre- quency of gus positive cells was observed to be 10 –4 . It has been observed that the silicon-carbide fibres meticulously mediated the exact delivery of DNA right into the cytoplasm as well as the nuclei of cells in a manner very much akin to microinjection.
Applicability of this particular technique was successfully extended to both maize and tobacco suspension culture cells. Unfortunately, it has not yet been well substantiated and established whether the transformation(s) thus accomplished give rise to fairly stable integrations of the transgene.
* Light amplification by stimulated emission of radiation that emits intense heat and power of close range. ** The infection of bacteria by purified phage DNA after pretreatment with Ca 2+ ions or conversion to
spheroplasts.
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2.3.11. Transformation by Ultrasonication
In several vegetative species wheat, tobacco and sugarbeet explants after being cultured for a certain duration were adequately sonicated with plasmid DNA that essentially carried marker genes e.g., cat, nptil and gus. It has been observed that when the sonicated calli were carefully transferred to selective medium-it gave rise to adequate shoots ; whereas all such controlled calli (not sonicated with plasmid DNA) proved to be 100% fatal.
Example : Tobacco : In this specific instance the transgenic plants were duly obtained at an approximate frequently of 22 per cent.*