Indian Journal of Bioteechnology Vol 6, January 2007, pp 31-34

Standardization of different parameters for particle gun mediated genetic transformation of sugarcane (Saccharum officinarum L.)
Ajinder Kaur, M S Gill, Raman Gill and S S Gosal*
Department of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana 141 004, India Received 18 March 2005 ; revised 13 January 2006 ; accepted 28 March 2006 Three different parameters, viz. quantity of DNA, size of target tissue and distance between microcarrier launch assembly and target tissue (firing distance), for particle gun (Bio-Rad) mediated transformation were optimized using embryogenic calli of sugarcane (target tissue), gusA (reporter gene) and hpt (hygromycin resistance gene). Of 12, 18 and 24 L of DNA (gusA and hpt) suspension used per two bombardments, highest (33.33%) GUS expression was observed with 18 L of suspension carrying 1.88 g DNA. Bombardments made into 2-5 mm calli exhibited 55.55% GUS expression, while no expression was obtained in 7.5-12 mm calli after 7 h of their incubation in X-gluc. Out of different firing distances, viz. 2.5, 5.0, 7.5 and 10.0 cm, maximum intensity of GUS expression was recorded when calli were bombarded at a distance of 5.0 cm from the microcarrier launch assembly in the particle gun. Keywords: embryogenic calli, GUS assay, particle gun bombardment, sugarcane, transformation IPC Code: Int. Cl.8 A01H5/00; C12N15/9

Introduction Among the various direct gene transfer methods, particle gun or shot gun bombardment is the most commonly and widely used method for plant transformation. This method of gene transfer is rapid, efficient, tissue non-specific and can be employed for simultaneously bombarding more than one gene (co-transformation) in a host plant. As many as 14 genes have been co-introduced in rice by this method1. Using particle gun bombardment of embryogenic calli, transgenic plants of sugarcane were first developed in Australia2. Since then, it has been used by several workers to produce transgenics in sugarcane3,4,5. However, reports are not available on successful transformation of Indian sugarcane varieties using particle gun for developing resistance against insects and diseases. Particle bombardment is a physical method of transformation in which DNA is coated on microscopic (0.2-0.7 m) heavy metal (gold/tungsten) particles. These particles are then accelerated to high velocity and bombarded into cells. Several factors, like kind and concentration of selective agent,
__________________ *Author for correspondence: Tel: 91-161-2401960-270 (Extn.); Fax: 91-161-2400945 E-mail: ssgosal@rediffmail.com

genotype of target tissue, quantity of DNA, size of the target tissue, distance between microcarrier launch assembly and target tissue, rupture disc pressure employed, etc., affect the frequency of transformation mediated by particle gun. Keeping all this in view, experiments were carried out to standardize important factors, such as quantity of DNA, size of target tissue and distance between microcarrier launch assembly and target tissue, using a commercially grown sugarcane variety CoJ 83. Materials and Methods Embryogenic calli of sugarcane variety CoJ 83 were used as target tissue for particle gun (Bio-Rad USA) bombardment. Various accessories of the gun included microcarriers or tungsten particles of size 0.6 m, rupture discs for pressure 1100 psi, rupture disc holders, macrocarriers of thickness 51 m, macrocarrier holders, macrocarrier launch assembly, shelf for placing the target tissue and a helium gas cylinder to provide the required pressure. Plasmid DNA carrying the gene construct (gusA and hpt with maize ubiquitin promoter) was used for transforming calli. gusA is a reporter gene and hpt is a selectable marker gene, which imparts resistance to the antibiotic hygromycin B. Prior to carrying out transformation, a suspension of tungsten particles was prepared and then DNA was coated on

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this suspension following the protocols described below.

Protocol for Preparing Suspension of Tungsten Particles

1. Put 30 mg of tungsten particles in 1.5 mL eppendorf tube. 2. Add 0.5 mL of absolute alcohol and vortex for 1-2 min. 3. Centrifuge for 1 min at 10,000 rpm and pipette off the supernatant. 4. Repeat steps 2 and 3 twice. 5. Add 0.5 mL of sterile water, resuspend, centrifuge and remove the supernatant. Repeat 3 times. 6. Resuspend microcarriers in 0.5 mL of sterile distilled water. 7. Aliquot 50 L of the final suspension into 1.5 mL eppendorf tubes. 8. Store at 4C or room temperature.
Protocol for Coating DNA on Tungsten Particles

11. Install macrocarriers into macrocarrier holders. 12. Pipette 9 L of the DNA coated microcarriers onto the centre of each macrocarrier. (Each preparation is sufficient for 5 bombardments) 13. Dry for about 1 min and use for bombarding calli. Bombarded calli were placed on recovery medium MS6 + 2,4-D (2.5 mg L-1) + Kn (0.5 mg L-1) + sucrose (30 g L-1) for 2-3 d and then subjected to histochemical GUS assay. Appearance of blue sections in the calli was taken as an indicator of gusA expression in the bombarded calli. Results and Discussion
Quantity of DNA for Bombardment of Embryogenic Calli through Particle Gun

1. Under continuous vortexing, add 50 L tungsten particle suspension and the following in order given below: 5 L DNA (DNA concentration = 1 g/L), 50 L CaCl2 (2.5 M; autoclaved) and 20 L spermidine (0.1 M). 2. Continue vortexing for 3 min. 3. Allow microcarriers to settle for 1 min. 4. Spin at 7,000 rpm for 1.0 min. 5. Remove supernatant. 6. Add 140 L of 70% ethanol without disrupting the pellet. 7. Remove supernatant. 8. Add 140 L of 100% ethanol without disrupting the pellet. 9. Remove supernatant. 10. Resuspend microcarriers in 48 L of 100% ethanol.

Tungsten particles were used for bombarding DNA into plant cells because they are inert in nature and do not react with DNA, other chemicals used in the preparation of suspension and plant metabolites. Three different quantities of DNA (gusA and hpt) coated tungsten particle suspension, viz. 6, 9 and 12 L were loaded on three macrocarriers and bombarded into embryogenic calli of sugarcane variety CoJ 83. In each case, two bombardments after an interval of 4 h were made into the calli, i.e. the calli were transformed with total volume of 12, 18 and 24 L of DNA suspensions. Multiple bombardments allow better coverage of the target area and compensate for misfires from faulty and poorly set rupture discs7, and hence increase the frequency of transformation of target tissues. The amount of DNA and tungsten particles contained in different quantities of the suspension is given in Table 1. A total of 24, 30 and 26 calli were bombarded with 12, 18 and 24 L of DNA suspension, respectively. After 3 d of bombardment, GUS assay of bombarded calli was

Table 1Percentage and intensity of GUS expression in calli of sugarcane variety CoJ 83 bombarded with different quantities of DNA suspension (after 20 h of incubation in X-gluc) Amount of DNA suspension used/two shots 12 L (1.25 g DNA, 0.75 mg TPs)* 18 L (1.88 g DNA, 1.13 mg TPs) 24 L (2.50 g DNA, 1.50 mg TPs) Total no. of calli bombarded 24 30 26 No. of calli incubated in X-gluc 8 6 10 No. of calli exhibiting GUS expression 2 2 2 % GUS expression and intensity 25.00 (Light blue) 33.33 (Medium blue) 20.00 (Medium blue)

*Figures in parentheses depict amount of DNA and tungsten particles (TPs) contained in the suspension

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performed, which revealed blue expression in all calli groups. About 25, 33.33 and 20% expression was recorded in calli bombarded with 12, 18 and 24 L of suspension, respectively (Table 1). gusA is a reporter gene that encodes for -glucuronidase enzyme and acts on the substrate X-gluc, producing blue colour in the transgenic tissues. GUS expression was first observed in calli bombarded with 24 L DNA suspension, i.e. after 4 h of calli incubation in X-gluc. However, further incubation, even up to 20 h, did not increase the percentage of calli exhibiting GUS expression. In the calli bombarded with 18 L suspension, light blue expression was observed after 5.45 h of incubation. In this case, number of calli exhibiting GUS expression increased with increase in period of incubation. While in calli bombarded with 12 L suspension, GUS expression was seen only after 20 h of incubation. Thus, 18 L of DNA suspension/two bombardments carrying 1.88 g of DNA and 1.13 mg of tungsten particles was found optimum for carrying out particle gun mediated transformation of sugarcane embryogenic calli. In Arabidopsis, tobacco and birch, optimal amount of DNA for transient expression of gusA and luc (firefly luciferase) gene construct using HeliosTM Gene Gun was worked out to be 2 g/two shots and the best particle size was 0.6 m for all targets8. Bombardment with same amount of DNA produced highest number of GUS cells/leaf in case of wheat9.
Size of Target Tissue

Table 2Percent GUS expression in different size calli of sugarcane variety CoJ 83 bombarded with 18 L of DNA suspension Size of calli Total no. of No. of calli % GUS Time calli incubated exhibiting expression taken for in X-gluc GUS expression expression 2-5 mm 7.5-12 mm 7.30 h No expression even after 48 h Table 3Intensity of GUS expression in calli of sugarcane variety CoJ 83 bombarded (with 18 L of DNA suspension) at different distances from the microcarrier launch assembly 9 5 5 0 55.55 0

Firing distance Intensity of GUS Improvement in (cm) expression in calli after 7 h intensity after 13.30 h of incubation in X-gluc of incubation 2.5 5.0 7.5 10.0 Very-very light Light Very-very light Very-very light No Yes Yes No

Two different sized calli of CoJ 83 measuring 2-5 mm and 7.5-12 mm were used as targets for bombardment with 18 L of DNA suspension. A total of 20 calli of each size were bombarded. It was observed that 2-5 mm size calli exhibited 55.55% GUS expression after 7.30 h of their incubation in X-gluc; whereas, 7.5-12 mm calli did not develop blue colour even after 48 h of their incubation (Table 2). Therefore, embryogenic calli of 2-5 mm size should be used for effective transformation through particle gun in sugarcane.
Firing Distance (Distance between Microcarrier Launch Assembly and Target Tissue in the Particle Gun)

In this experiment, four firing distances were tried by placing the target calli (2-5 mm size) at 2.5, 5.0, 7.5 and 10.0 cm distance from the microcarrier launch assembly in the particle gun, and bombarded with 18 L of DNA suspension. It was observed that the calli bombarded at a distance of 5.0 cm from the

assembly exhibited light GUS expression, whereas calli placed at other positions exhibited very light expression after 7 h of their incubation in X-gluc (Table 3). Intensity of blue colour increased in the calli bombarded at 5.0 cm and 7.5 cm distance after 13.30 h of incubation. Further, the improvement in colour was better in calli kept at 5.0 cm distance than in calli bombarded at 7.5 cm distance. But GUS expression in calli bombarded at 2.5 cm and 10.0 cm distance did not increase with increase in incubation. Also, number of calli (bombarded at a distance of 10.0 cm distance) exhibiting GUS expression did not increase with time. So, the target calli should be kept at 5.0 cm distance from microcarrier launch assembly in the particle gun for efficient transformation. Best transformation results were obtained at a pressure of 1100 psi and a target distance of 6.0 cm when embryonic axes of Vigna unguiculata were bombarded with gusA and bar genes7. In case of rose cultivar Glad Tidings, the level of GUS expression in embryogenic callus was consistently greater when a firing distance of 7.0 cm was used as compared to a distance of 10.0 cm, irrespective of the rupture disc pressure employed10. In conclusion, bombardments made with 18 L of DNA suspension into 2-5 mm size calli, placed at 5.0 cm distance from the microcarrier launch assembly in the particle gun, were optimum for achieving higher transformation efficiency in sugarcane.

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Acknowledgement The financial assistance from the Department of Biotechnology (DBT), Government of India, New Delhi is gratefully acknowledged. References
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5 Falco M C, Tulmann Neto A & Ulian E C, Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane, Plant Cell Rep, 19 (2000) 1188-1194. 6 Murashige T and Skoog F, A revised medium for rapid growth and bioassays with tobacco culture, Plant Physiol, 15 (1962) 473-497. 7 Ikea J, Ingelbrecht I, Uwaifo A & Thottappilly G, Stable gene transformation in cowpea (Vigna unguiculata L. walp.) using particle gun method, African J Biotechnol, 2 (2003) 211-218. 8 Helenius E, Boijie M, Niklander-Teeri V, Tapio Palva E & Teeri T H, Gene delivery into intact cells using the HeliosTM Gene Gun, Plant Mol Biol Rep, 18 (2000) 2870-2871. 9 Schweizer P, Pokorny J, Abderhalden O & Dudler R, A transient assay system for the functional assessment of defense-related genes in wheat, MPMI, 12 (1999) 647-654. 10 Marchant R, Power J B, Lucas J A & Davey M R, Biolistic transformation of rose (Rosa hybrida L.), Ann Bot, 81 (1998) 109-114.