INTRODUCTION

The term ‘direct gene transfer’ is used to discriminate between methods of plant transformation that rely on the use of Agrobacterium (indirect methods) and those do not (direct methods) [2]. There are several methods for direct gene transformation such as electroporation, PEG mediated transformation, cationic carriers and biolistics. Particle bombardment (‘biolistics’) is a physical method widely used for gene transfer into plants, mammals, fungi and bacteria (Armaleo et al. 1990 [4], Klein et al. 1992 [5], Shark et al. 1991 [6], Williams et al. 1991[7], Zelenin et al. 1989[8]) [3]. In this technique, the DNA constructs coated with Gold particles are delivered at a high velocity into the target which is two plants here (Arabopsis thaliana and Nicotina tobacum). And the DNA constructs are pUC19 (contol plasmid without P358-GUS) as a negative control and pIG 121-HM which has reporter gene GUS encoding ß-Glucoronidase (GUS enzyme). This enzyme uses X-gluc substrate and gives blue color end product, indicating the success of transformation.

MATERIALS AND METHODS

Plant Material

Arabopsis thaliana plants were grown in peat at 20oC with an 8 hour photoperiod. The 4 to 5 weeks old plants were used for gene delivery bombardment experiments and Nicotiana tabacum (tobacco) plants were grown at 23oC with a 16 hour photoperiod in vermiculite. When the first true leaf expanded, the plants were used for the bombardment.

 

Preparation of DNA-coated gold microcarriers for the Gene Gun system

5 mg gold microcarriers (diameter: 0.6 mm) and 10 ml of 0.05 M spermidine were vortexed for 5 s and sonicated for 10 s in an ultrasonic bath to break up gold clumps. Secondly, 10 mg plasmid DNA (pUC19 or pIG121 Hm) in 10 ml H2O is added. Then, 10 ml 1 M CaCl2 was added dropwise while vortexing the mixture at moderate rate. The mixture was incubated for 10 min at room temperature to precipitate the gold and DNA. After centrifugation for 15 s at 14,000 rpm, the pellet was washed 3 times in 0.1 ml absolute ethanol. The pellet was resuspended in 20 ml ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP). The final volume was adjusted to 0.6 ml using the ethanol/PVP solution. The suspension was then used for the cartridge preparation as follows.

Catridge preparation with the Tubing Prep Station for the Gene Gun system

Firstly, the Gold-Coat tubing was first dried by purging with nitrogen for 15 min. Then, the suspension was drawn into the Gold-Coat tubing (5” in length) and placed into the Tubing Prep Station following, microcarriers are allowed to settle for 10 min. Then ethanol then was removed slowly. The Gold-Coat tubing was rotated and the particles were spread onto the inner surface of the tubing and subsequently dried with a flow of nitrogen. Any unevenly coated sections should be discarded before the remaining tubing was cut into 0.5” pieces with the Tubing Cutter. Eight cartridges were generated.

Preparation of DNA-coated gold microcarriers for the PDS-1000/He system

Stock suspension of microprojectiles was prepared by mixing 60 mg of 1.0-mm gold particles in 1,000 ml of absolute ethanol which can be stored at -20 oC. Then stock suspension was vortexed for 30 s and soon after 35 ml of the stock suspension was quickly removed and added to a 1.5 ml microcentrifuge tube to be microcentrifuged at high speed for 30 s. After removing ethanol with micropipette, pellet was resuspended in 1 ml water and microcentrifuged for 5 min. Then, microprojectiles were resuspended in 25 ml of DNA solution (pUC19 or pIG121-Hm) (1 mg/ ml). After that these reagents were added in written order and at the amount specified: 220 ml of water, 250 ml of 2.5 M CaCl2, and 50 ml of 0.1 M spermidine (Sigma Chemical S-0266 stock solution stored at –20 oC, diluted 14 ml to 1,000 ml with water). Later on, the solution was mixed thoroughly and vortexed on a vortex shaker for at least 10 min at 4 oC and microcentrifuged for 5 min and remove supernatant. Resuspension of DNA/microprojectile precipitate in 600 ml of absolute ethanol by pipetting up and down several times was the next step before another microcentrifugation for 1 min. Removal of ethanol and resuspension of pellet in 36 ml of absolute ethanol by pipetting up and down until well dispersed then were done before loading to the macrocarrier sheet. Lastly, 10 ml of the suspension was pipetted as evenly as possible onto the center of a Mylar macrocarrier sheet. It is important to let the ethanol evaporate.

 

Each DNA/microprojectile suspension yields enough solution to coat 3 Mylar macrocarriers.

Bombardment Conditions and Transient Gene Expression
Intact leaves of Arabidopsis and tobacco plants were bombarded with the Gene Gun system. The helium pressure for Arabidopsis was 75psi

Detached Arabidopsis leaves were bombarded with the PDS-1000/He unit. First, 1,100 psi rupture disk (that has been soaked with isopropanol) was placed in the unit. The distance between the rupture disk and macrocarrier was 8-10 mm and between macrocarrier and stopping screen is 1 cm. Each Petri dish containing the leaves was placed in the device 6 cm below the stopping screen. Then chamber was evacuated to 28-29 mm Hg before bombarding the target.

Storage procedure

After bombardment, the leaves were sprayed with water and placed in a growth chamber for 24 hours before assaying for the GUS enzymatic activities. The detached leaves were incubated in a moisturized container.

 

Histochemical GUS assay
The leaves were submerged in 1 mM 5-bromo-4-chloro-indolyl-b-D-glucuronide (X-Gluc) in a buffered solution [100 mM Na-phosphate buffer (pH 7.0), 10 mM EDTA, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 1.0 mM X-Gluc, 0,1% Triton X-100] and incubated in the dark for 16 hours at 37 oC.

After staining, chlorophyll was removed with absolute ethanol in order to better visualize the blue spots.

RESULTS:

Figure 1: Gene gun (pIG121 Hm)

Figure 2: Gene gun (pUC19)

Figure 3: PDS-1000/He (pIG121 Hm)

Figure 4: PDS-1000/He (pUC19)

 

If expressed, ß-Glucoronidase enzyme uses X-Gluc and gives a blue colored and product thus indicating the succesfull transformation and expression. pIG121Hm carries the gene and promoter whereas, pUC19 does not (negative control).

DISCUSSION

Despite, Argobacterium based based transformation widely used; it is almost but exclusively restricted to dicotyledons [9] Since, monocotyledonous plants, which constitute some of the world's most important food crops, there is an alternative method is required. Microprojectile bombardment, which is non-specific, provides an ideal alternative approach and has been successfully employed to transform several of the major cereals, including barley maize wheat rice together with other monocotyledons such as tulip and orchids [13].Unlike the other direct transformation methods, the Microprojectile bombardment permits the delivery of DNA to the wide range of plants (review), making it the choice of method used in transformation.

 

There are several parameters that affect the efficiency of this method. These include the properties of microprojectile, tissue culture method of the plants, DNA construct and etc.

Nature of Microprojectiles

The two major microprojectiles or microcarriers are gold and tungsten particles. The size of the particle is important and should be optimized in such a way that can deliver significant amount of DNA without causing damage. Beside this the spherical shape is advantageous as it gives less damage to the cell. In general, tungsten particles have an irregular surface and are predisposed to agglomeration during the DNA coating procedure, whereas gold microprojectiles, which are nearly spherical, remain separated [10].Moreover, these particles should be inert materials otherwise they may cause toxicity and degradation of DNA in the cell. The gold particles are not toxic to any of the cells assessed by Sanford et al. [11] and did not catalytically attack DNA adsorbed to the surface of particles where as, tungsten is toxic to certain cell types and is also subject to surface oxidation, which affects DNA binding and degradation of adhered DNA[13]. Finally, the concentrations of these carriers are another parameter to be considered. If the concentration too

Low, it will give sparse coverage over the target area while high concentrations of microprojectiles can result in agglomeration of the particles during the DNA coating procedure. Agglomerated particles can lead to excessive damage to plant tissues upon bombardment

Adherence of DNA to microprojectiles

No doubt that the efficient DNA coatings of microprojectiles are essential for optimal delivery to the target. In both two procedures CaCl2 was used to precipitate DNA with gold particles. Polyvinyllpyrrolidone (PVP) was used as an adhesive during the coating of DNA/microcarrier suspension to the walls of the Goldcoat tubing[bulletin], resulting increased evenly coating. And the spermidine which is a polyamide was used to increase static charges on plasmid GC rich regions hence increasing the coating efficiency. Sonification was done to disrupt any aggregated particles. Resuspension of DNA/microprojectile with ethanol permits the precipitate to dry as ethanol evaporates easily.

Uneven precipitation of DNA and the aggregation of microprojectiles are the problems encountered during the coating of DNA/microprojectiles. Uneven precipitation is due to rapid process of precipitation itself. The vortexing procedure is commonly used while adding the calcium chloride drop wise to the DNA microprojectile mixture.

Increasing the concentration of DNA used to coat the microprojectiles should, theoretically, increase the transformation frequency in a linear manner, until saturation of the microprojectiles is achieved. However, high concentrations of DNA have been shown to result in agglutination of the

 

microprojectiles, which reduce transformation frequencies because of the large size and, effectively, the reduced number of particles [12].

Delivery of Microprojectiles into Target Tissues

Accelerating power, distance between device and the target and the vacuum in PDS-100/He system are important factors for velocity and so for penetration. Most systems utilize a vacuum to reduce air drag on the microprojectiles and to reduce the risk of shock waves, from the overlying gas, damaging the tissue [11]. Vacuum mediates a uniform distribution of particles.

Incorporation of Introduced DNA into the Recipient Genome

Integration of the DNA construct into the chromosome results in stable expression of the transgene where as otherwise, it will give a transient expression or most probably nothing. In a clearer sentence, the gene expression is related to the ultimate location of microprojectiles within cells.

Choice of DNA Construct

The choice is extremely dependent on the purpose of the experiment itself. In this study, the purpose was to observe the succession of the two different gene bombardment procedures by using a reporter enzyme GUS. The GUS enzyme is encoded by an E coli gene (gusA or udiA). GUS (ß-Glucoronidase) cleaves a colourless substrate, X-Gluc (5 bromo-4-chlom-3-indolyl-l~-D-glucoronic acid) into a product which, on oxidation, produces an indigo coloured dye. Hence, the transformed cells can be identified by their blue colouration [13].

The DNA construct pIG121Hm that was used contains this enzyme with a promoter called Cauliflower Mosaic Virus (CaMV) 35S promoter where as the DNA construct pUC19 does not contain the gene, used for negative control. The CaMV 35S promoter is very strong and seemed to be constitutive, meaning inducing the expression of the downstream located coding region, in apparently all plant tissues [1]

Configuration of the vector used for gene delivery may influence gene integration and expression as linear forms of plasmid resulted in higher levels of gene expression than supercoiled DNA [13].

Constructs with selectable marker which is used to select the transformed cells to get a new plant can be used with a transgene of interest. The selectable marker and transgene may have their own promoters one is constitutive and the other is development or tissue specific depends on the interest opening an amazing doors of the science for use of the humans.

Choice and Preparation of Explants

In the experiment the leaves used were young allowing easy penetration for the bombardment.

 

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3. http:// www.bio-rad.com/gene transfer/ , Technical Bulletin 2351

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5. Klein R. M., et al., High-velocity microprojectiles for delivering nucleic acids into living cells, BioTechnology, 24, 384-386 (1992)

6. Shark, K. B., et al., Biolistic transformation of a procaryote, Bacillus megaterium, Appl. Environ. Microbiol., 57, 480–485 (1991)

7. Williams, R. S., et al., Introduction of foreign genes into tissues of living mice by DNA-coated micro p rojectiles, Proc. Natl. Acad. Sci. USA, 8 8, 2726–2730 (1991)

8. Zelenin A. V., et al., Genetic transformation of mouse cultured cells with the help of high-velocity mechanical DNA injection, FEBS Lett., 244, 65–67 (1989)

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10. Hunold, R., Bronner, R. and Hahne, G., Early events in microprojectile bombardment: cell viability and particle location. The Plant J., 5,' 593-604 (1994)

11. Sanford, J.V, Smith, F.D. and Russell, J.A. Optimising the biolistic process for different biological applications. Methods Enzymol., 217, 483-509 (1993).

12. Oard, J.H., Physical methods for the transformation of plant Cells, Biotech. Adv., 9, 1-I 1 (1991)

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