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Plant Cell Tiss Organ Cult (2012) 109:401–410 DOI 10.1007/s11240-011-0104-7

ORIGINAL PAPER

Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn Swati Jagga-Chugh • Sumita Kachhwaha Manju Sharma • Aditi Kothari-Chajer • S. L. Kothari



Received: 24 June 2011 / Accepted: 23 December 2011 / Published online: 8 January 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Microprojectile bombardment mediated genetic transformation parameters have been standardized for seed derived callus of Eleusine coracana. Plasmid pCAMBIA 1381 harboring hygromycin phosphotransferase (hptII) as selectable marker gene and b-glucuronidase (gus A) as reporter gene, was used for the optimization of gene transfer conditions. The transient GUS expression and survival of putative transformants were taken into consideration for the assessment of parameters. Optimum conditions for the microprojectile bombardment mediated genetic transformation of finger millet were 1,100 psi rupture disk pressure with 3 cm distance from rupture disk to macrocarrier and 12 cm microprojectile travel distance. Double bombardment with gold particles of 1.0 lm size provided maximum transient GUS expression and transformation efficiency. Osmotic treatment of callus with 0.4 M sorbitol enhanced efficiency of particle bombardment mediated genetic transformation. Regenerative calli were bombarded at optimum conditions of bombardment and placed on regeneration medium with hygromycin to obtain transformed plants. The integration of hptII and gus A genes was confirmed with PCR amplification of 684 and 634 bp sizes of the bands respectively from putative transformants and Southern blot hybridization using PCR amplified DIG labeled hptII gene as probe. PCR analysis with hptII gene specific primers indicated the presence of S. Jagga-Chugh  S. Kachhwaha  S. L. Kothari (&) Centre for Converging Technologies, University of Rajasthan, Jaipur 302 004, India e-mail: [email protected]; [email protected] S. Kachhwaha  M. Sharma  A. Kothari-Chajer  S. L. Kothari Experimental Morphogenesis and Plant Tissue Culture Laboratory, Department of Botany, University of Rajasthan, Jaipur 302 004, India

transgene in T1 generation plants. Thus a successful genetic transformation system was developed using particle bombardment in E. coracana with 45.3% transformation efficiency. The protocol will be helpful for the introgression of desired genes into E. coracana. Keywords Finger millet  Microprojectile  Genetic transformation  Regeneration  Southern blot hybridization  Transgenic plants Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid GA3 Gibberellic acid GUS b-Glucuronidase hptII Hygromycin phosphotransferase MS Murashige and Skoog medium

Introduction Particle bombardment mediated genetic transformation provides an alternative method of gene transfer in those cases where other methods of gene transfer are not efficient. It facilitates DNA delivery into intact plant cells, simultaneous multiple gene transfers with no biological constraints or host limitations (Altpeter et al. 2005). Moreover, particle bombardment is also employed for DNA delivery in transient gene expression studies to investigate the plant gene expression and for its ability to introduce DNA directly into different tissues (Sivamani et al. 2009). Any kind of plant tissue could be used as an explant for microprojectile bombardment, which is an advantage over other methods. The common target tissue for particle bombardment is embryogenic somatic tissues such as immature embryo, isolated scutella, inflorescence

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and regenerative callus derived from such tissues (Christou 1997). Shoot-apex was also used as an explant to particle bombardment for production of transgenic cotton with an increased level of phytase activity (Liu et al. 2011). Particle gun has been used for transformation of all the major cereals and millets (Tama´s et al. 2009; Um et al. 2007; Ogawa et al. 2008; Girgi et al. 2006; Latha et al. 2006; James et al. 2008) including finger millet (Latha et al. 2005). Although Agrobacterium mediated gene transfer in finger millet has been reported earlier by the authors (Sharma et al. 2011), however, in the present study we have reported the feasibility of direct DNA delivery to seed derived callus by particle bombardment and generation of highly efficient, reproducible transformation protocol for finger millet. Finger millet (Eleusine coracana (L.) Gaertn.) is also known as African millet. It has remarkable attributes as a subsistence food crop which can survive in poor soils under harsh and severe drought conditions (Latha et al. 2005). Finger millet is consumed as a whole meal, which has rather high crude fiber content (3–4%). Its seed can be stored for several years without any insect damage. Increase in crop yield in future is likely to come from improved varieties transgenetically modified for resistance to abiotic and biotic stress, using a tertiary gene pool (Kothari et al. 2005). The major loss of finger millet yield is due to fungal infection, thus genetic engineering is desirable for production of fungal resistant finger millet. Particle bombardment parameters differ from one plant species to another and within a species from one cultivar to another. Development of microprojectile bombardment mediated transformation system will be helpful for the genetic improvement of this important millet crop.

Materials and methods Plant material and culture conditions Eleusine coracana PR-202 seeds were procured from the University of Agricultural Science, GKVK campus, Bangalore. Seeds were initially washed with 0.5% (v/v) Tween20 and left in 70% (v/v) ethanol for 5 min, before surface sterilization with 2.5% sodium hypochlorite solution (v/v) for 10 min followed by thorough washing with autoclaved distilled water. Seeds were placed on callus induction medium, i.e., MS medium (Murashige and Skoog 1962) supplemented with 0.5 mg l-1 Kinetin and 2 mg l-1 2,4-D. After 6 weeks of culture green nodular callus was obtained and further maintained on medium containing 0.2 mg l-1 2,4-D by repeated subculture in every 3 weeks (KothariChajer et al. 2008). Nodular callus from 2nd and 3rd subculture was used for bombardment. The bombarded callus

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was transferred to regeneration medium supplemented with 1 mg l-1 GA3. MS basal medium was supplemented with 3% (w/v) sucrose and solidified with 0.85% (w/v) agar (bacteriological grade, Himedia, Mumbai, India). Cultures were incubated at 26 ± 1°C under fluorescent light (75 lmol s-1 m-2) and 16/8 h photoperiod. Transformation with particle gun Green nodular calli (5–7 mm) were arranged aseptically in a circle with diameter of 20 mm in Petri plates on MS medium supplemented with 0.2 mg l-1 2,4-D and 0.4 M sorbitol 4 h prior to bombardment. Plasmid pCAMBIA 1381 was isolated using plasmid Miniprep kit (Fermentas, India) following manufacturer’s protocol. Transformation conditions were determined using the plasmid pCAMBIA 1381, which harbours gus A reporter gene and the hptII gene as selectable marker, both were controlled by the cauliflower mosaic virus (CaMV) 35S promoter. Plasmid DNA was concentrated according to Qiagen, India. Preparation of microcarriers Microparticles (6 mg) were suspended in 200 ll of 70% ethanol (v/v) by vigorous vortexing for 20 s to 1 min followed by soaking for 5–10 min. Then microparticles were washed with 100 ll of sterile water by vortexing and suspension was left at room temperature for 10 min followed by 1 min centrifugation at 10,000 rpm. After washing pellet was resuspended in 100 ll sterile 50% glycerol. Then 15 ll plasmid DNA (1 lg ll-1), 100 ll CaCl2 (2.5 M) and 40 ll cold spermidine (0.1 M) were added for coating of microparticles. After 10 min incubation on ice the suspension was spun in microcentrifuge for 30 s at 10,000 rpm then the supernatant was removed and pellet was washed with 70% (v/v) ethanol followed by washing with absolute ethanol. Then the DNA pellet was re-suspended in 60 ll absolute ethanol for bombardments. 5 ll of this suspension was loaded on macrocarrier and allowed to dry. Care was taken to ensure uniform particle distribution and minimize agglomeration. To prevent agglomeration microcarrier particle suspension was vortexed prior to each bombardment. Microprojectile bombardment Bombardments were carried out with biolistic gene gun (PDS 1000/He, Bio-Rad) under a vacuum of 25 inches of Hg. The variables to be optimized included, rupture disc pressures (450, 650, 900, 1,100 and 2,100 psi), distance from rupture disk to macrocarrier (3, 6 and 9 cm) and microprojectile travel distances (6, 9, and 12 cm), type of microcarrier (gold and tungsten), size of microcarrier (0.6,

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1.0 and 1.6 lm diameter of gold particles), number of bombardments per Petri plate (1, 2 and 3), type of osmoticum (sorbitol, mannitol and sucrose) and concentration of osmoticum (0, 0.2, 0.4 and 0.6 Molar). These factors were standardized stepwise and conditions optimized at one step were used in following step, which finally resulted into development of efficient system for production of transgenic finger millet. Calli bombarded with uncoated microcarriers were used as control. Selection and regeneration of putative transformants After bombardment, calli were kept on medium supplemented with 0.2 mg l-1 2,4-D and 0.4 M sorbitol at 25°C for 18 h and then transferred to medium supplemented with 0.2 mg l-1 2,4-D without antibiotic. After 15 days calli were transferred to selection medium i.e., MS medium containing 0.2 mg l-1 2,4-D, 3% sucrose, 0.85% agar and hygromycin. After 3 cycles of selection with increasing concentration of hygromycin (10, 20 and 50 mg l-1), green calli were transferred to regeneration medium supplemented with 1 mg l-1 GA3 and 50 mg l-1 hygromycin. In vitro regenerated putative transformants were transferred under controlled green house conditions and mature seeds were collected. In order to obtain T1 progenies mature seeds from T0 hptII positive plants were randomly selected and germinated on ‘ MS medium supplemented with 50 mg l-1 hygromycin. DNA was extracted from seedling and amplified with hptII gene specific primers.

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TTAACCACAAACC 30 and R: 50 GCCAGAAGTTCTT TTTCCAGTACC 30 ). These primers were obtained from Banglore Genei, India. PCR amplified hptII gene and gus A gene present in pCAMBIA 1381 plasmid were taken as positive control. DNA from nontransformed plants was used as negative control. Genomic DNA (approx. 40 ng) along with 1.5 mM MgCl2 and 50 ng of hptII/gus A gene forward and reverse primers in 25 ll volume was subjected to PCR amplification (Bio-Rad, UK), using an initial denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 45 s, 52°C for 45 s and 72°C for 45 s, and a final extension step at 72°C for 7 min. The amplified products were separated on 1.2% agarose (Himedia, Mumbai, India) gel and photographed using Gel Documentation System (Bio-Rad, UK). Southern blot hybridization Approximately 8–10 lg genomic DNA of transformed plants was digested with BamH1at 37°C. PCR amplified hptII gene served as a positive control and DNA from nontransformed plant was used as negative control. It was electrophoresed on 0.8% agarose gel and blotted on immobilon-NY ? membrane (Millipore, India; cat. no. 7104 633) as per Sambrook and Russel (2001). PCR amplified hptII gene, labeled with digoxigenin (DIG) dUTP was used as probe. Blotted membrane was hybridized with probe using the standard protocol provided in DIG High prime DNA labeling and detection starter kit I (Roche; cat. no. 11 745 832 910) for color detection with NBT/BCIP.

Histochemical GUS assay Statistical analysis GUS assay (Jefferson et al. 1987) was performed for calli and leaves of putative transformants by placing them in Eppendorf tubes containing GUS-buffer (1 mM 5-bromo, 4-chloro, 3 indolyl-D-glucuronide (XGluc), 100 mM sodium phosphate buffer pH 7.0, 0.5 mM potassium ferricynide, 0.5 mM potassium ferrocynide, and 0.1% Triton X-100). Tubes were incubated overnight at 37°C, washed once with sterile distilled water and finally dipped in 70% ethanol overnight to extract any chlorophyll that may be present in the tissue. Leaf and callus were examined under a dissecting microscope and scored for blue coloration.

Each treatment consisted of at least three plates and was replicated thrice. Percent GUS activity was calculated as number of calli showing GUS expression to the total number of explants stained after bombardment. Transformation efficiency was evaluated as the number of plants showing hptII gene amplification per total number of bombarded calli. Analysis for variance (ANOVA) appropriate for the design was carried out to detect significant difference among different means, which were compared using Duncan’s multiple range test at the 5% probability level according to Gomez and Gomez (1984).

PCR amplification DNA was isolated from putative transformants and control shoots using 100 mg of in vitro grown leaves with Qiagen mini DNA kit (Genetix). Transformation was confirmed by PCR with hptII (hygromycin selection marker gene) specific primers (F: 50 GCTCCATACAAGCCAACCAC 30 and R: 50 CGAAAAGTTCGACAGCGTCTC 30 ) and gus A (reporter gene) specific primer (F: 50 AACAGTTCCTGA

Results Factors influencing particle gun mediated DNA delivery including rupture disk pressure, distance from rupture disk to macrocarrier and microprojectile travel distance, type of microcarrier, size of microcarrier, number of bombardments per Petri plate, types of osmoticum and concentration of

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osmoticum were standardized for finger millet embryogenic callus.

1.0 lm gave the highest 87.6% GUS expression as well as (35.2%) transformation efficiency (Fig. 1e; Table 1).

Effect of rupture disk pressure

Effect of number of bombardments per Petri plate

Different helium pressures were found to affect transient GUS expression as well as transformation efficiency. When callus was bombarded using various rupture disk pressure with 3 cm distance between rupture disk and macrocarrier and 9 cm microprojectile travel distance, it was observed that 1,100 psi helium pressure gave highest transient GUS expression (83%) (Fig. 1a). The highest transformation efficiency (45.3%) was also observed when 1,100 psi rupture disk pressure was used (Table 1). Regeneration potential of callus was lost at 2,100 psi rupture disk pressure. Thus 1,100 psi pressure was used in further experiments for optimization of other factors.

Calli were bombarded single and multiple times to evaluate the effect of number of bombardments per Petri plate on transient GUS expression and efficiency of transformation. Double bombardment gave maximum transient GUS expression (78.6%) as compared to calli bombarded once and three times per Petri plate. The number of hptII gene positive plants as well as transformation efficiency (41.3%) was highest when callus was bombarded two times per Petri plate (Fig. 1f; Table 1). When callus was bombarded for three times per Petri plate necrosis was observed due to mechanical damage and plantlet regeneration per callus also decreased.

Effect of distance from rupture disk to macrocarrier and microprojectile travel distance

Effect of types and concentration of osmoticum

The effect of gap (3, 6 and 9 cm) between rupture disk and macrocarrier was recorded on transient GUS expression and transformation efficiency. Highest GUS expression (81.5%) and transformation efficiency (35.5%) was observed when 3 cm distance was used with 1,100 psi pressure and 9 cm microprojectile travel distance (Fig. 1b; Table 1). A marked decrease in percent explants showing GUS expression and transformation efficiency was observed beyond 3 cm distance from rupture disk to macrocarrier. The intensity of blue color and number of explants with blue color were also increased when microprojectile travel distance was increased. Optimum travel distance was observed to be 12 cm for highest GUS expression (82%) and efficiency of transformation (41.1%) (Fig. 1c; Table 1). While at 6 cm, distance from microcarrier and target tissue the transient GUS expression level (16%) and efficiency of transformation (7.7%) were lowest. Effect of type and size of microcarrier Tungsten microcarriers were also used to test their effect on transient GUS expression and transformation efficiency. It was observed that transient GUS expression (77.2%) and efficiency of transformation (22.8%) with gold particles were significantly higher than tungsten particles (Fig. 1d; Table 1). Use of tungsten particles adversely affected growth of calli as well as plantlet regeneration. Different gold microparticle sizes (0.6, 1.0 and 1.6 lm) were compared for their efficiencies in delivering DNA into the target tissues. Survival of calli and number of recovered putative transformants reached maximum when 1.0 lm size of gold particle was used (Table 1). Thus size of

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Significant difference was observed in percent explants showing GUS expression and transformation efficiency with different osmotic agents (sorbitol, mannitol and sucrose). Treatment of callus with sorbitol 4 h before and 18 h after the bombardment resulted into maximum transient GUS expression (81.3%) and efficiency of transformation (33.3%) (Fig. 1g; Table 1) whereas, the calli treated with sucrose and mannitol resulted in 16.1% and 21.9% transformation efficiency respectively. The effect of different concentrations of sorbitol (0, 0.2, 0.4 and 0.6 M) as osmotic treatment was also observed using the best conditions of other parameters described above. It was found that 0.4 M concentration of sorbitol significantly increased the level of transient GUS expression (76.6%) and transformation efficiency (29.5%) (Fig. 1h; Table 1). The concentration of osmoticum showing increased GUS expression in present investigation (0.4 M sorbitol) is considerably lower and thus may not have any adverse effect on regeneration. Callus survival as well as regeneration potential decreased when concentration higher than 0.4 M sorbitol was used. Selection, regeneration and histochemical GUS assay Calli were kept on the osmotic medium for 18 h following bombardment (Fig 2a) and transferred to selection medium after 15 days. Bombarded calli were transferred to medium with increasing concentration (10, 20 and 50 mg l-1) of hygromycin after every 15 days. During this selection process non-transformed calli turned brown gradually, while putative transformed sectors remained green and exhibited slow growth (Fig. 2b). After the three subcultures on selection medium, calli were transferred to regeneration

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Fig. 1 Effect of different factors on transient GUS expression in Eleusine coracana. a Effect of rupture disk pressure (psi); b effect of distance from rupture disk to macrocarrier (cm); c effect of microprojectile travel distance (cm); d effect of type of microcarrier; e effect of size of microcarrier (lM diameter); f effect of number of

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bombardment per Petri plate; g effect of type of osmoticum; h effect of concentration of osmoticum (Molar). Data represent the Mean ± SE followed by different letters are significantly different from each other at P = 0.05

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Table 1 Effect of different transformation conditions on transformation efficiency of finger millet Factors

No. of calli bombarded

No. of hygromycin resistant calli

No. of hygromycin resistant plants

No. of PCR (hptII) positive plants

Transformation efficiency (%)

Rupture disk pressure (psi) 450

75

21

11

10

13.3b

650

75

39

23

23

30.6c

900

75

48

26

24

32c

1,100

75

57

35

34

45.3d

2,100

75

6

0

0

32

32

35.5b

Distance from rupture disk to macrocarrier (cm) 3 90 69

0a

6

90

63

29

28

31.1b

9

90

21

5

4

4.4a

Microprojectile travel distance (cm) 6

90

42

9

7

7.7a

9

90

52

25

25

27.7b

12

90

66

38

37

41.1c

Gold

105

30

24

24

22.8b

Tungsten

105

24

16

16

15.2a

Type of microcarrier

Size of microcarrier (lM) 0.6

105

39

22

22

20.9a

1

105

53

37

37

35.2b

1.6

105

42

32

31

29.5b

30

13

12

16b

No. of bombardments per Petri plate 1 75 2

75

54

31

31

41.3c

3

75

24

7

7

9.3a

Sorbitol

105

54

36

35

33.3b

Sucrose

105

36

18

17

16.1a

Mannitol

105

30

23

23

21.9a

Type of osmoticum

Concentration of osmoticum (Molar) 0

105

36

19

19

18b

0.2

105

47

25

23

21.9b

0.4

105

48

31

31

29.5c

0.6

105

15

7

6

5.7a

Values in a column followed by different letters are significantly different from each other at P = 0.05

medium with 50 mg l-1 hygromycin. Shoot buds were induced on regeneration medium after 3 weeks of culture (Fig. 2c). White and green and only green well-developed plantlets regenerated from the same callus after 8 weeks of culture (Fig. 2d). Green plantlets were normal in appearance (Fig. 2e). Putative transformants were successfully established under controlled green house conditions (Fig. 2f). Meanwhile, calli and leaves of putative transformants were assayed for the transient GUS expression using nontransformed calli and leaves obtained from non transformed plants as control. The control callus and plant

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did not show any blue color (Fig. 2g, i) while GUS expression was observed in transformed tissues (Fig. 2h, j). Molecular analysis The genetic transformation was confirmed on the basis of expression of gus A reporter gene and selectable marker hptII gene. DNA of putative transformed shoots was subjected to standard conditions for PCR amplification with hptII and gus A gene-specific primers. Ten out of eleven transformants showed the expected 684 bp band size of

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Fig. 2 Microprojectile bombardment mediated genetic transformation, selection, regeneration and GUS assay in Eleusine using plasmid pCAMBIA 1381. a Callus cultured on medium supplemented with 2,4-D (0.2 mg l-1) ? 0.4 M sorbitol after bombardment; b Callus growth in selection medium (after 3 cycles of selection); c Shoot bud induction on regeneration medium supplemented with hygromycin (50 mg l-1) after 3 weeks of culture; d Regeneration of green and

albino plants on medium supplemented with hygromycin (50 mg l-1) after 8 weeks of culture; e Plantlet regenerated from bombarded callus; f Putative transformant established under control green house conditions; g GUS assay of control non-bombarded callus; h GUS expression in bombarded callus; i GUS assay of leaves of control plant; j GUS expression in leaves of putative transformant

hptII gene and eleven out of eleven expressed 634 bp bands of gus A gene amplified product in T0 generation (Fig. 3a, b). The DNA of control plant did not show any amplification. DNA of T0 transformants (hptII positive) was used for Southern blot hybridization with PCR amplified hptII fragment as a probe. The results indicated six out of ten selected plants with hptII gene integration in their genome and four samples of putative transformants with single copy while two transgenic plants contained two copies of the introduced gene (Fig. 4). There was no hybridization detected in the non-transformed plants. We observed (66.6%) single copy insertion of hptII gene by Southern blot analysis, confirming the efficacy of the present method. In T1 generation seven out of ten plants

were amplified with hptII gene specific primer (Fig. 5). The results revealed integration of hptII gene in T1 generation plants.

Discussion Particle bombardment has been widely exploited to produce tissues and plants expressing traits with agronomic value and has a major impact on basic plant science research and biotechnology (Altpeter et al. 2005; Taylor and Fauquet 2002). The objective of this work was to optimize different parameters in particle bombardment that could enhance stable integration of the desired genes in

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Fig. 3 a PCR amplification of hptII gene in putative transgenic T0 plants of Eleusine coracana. M molecular marker, PC positive control (684 bp band of hptII gene present in pCAMBIA-1381 plasmid), NC negative control (DNA from nontransformed plant), lanes 1–11 putative transformants. b PCR amplification of gus A gene in putative transgenic T0 plants of Eleusine coracana. M molecular marker, PC positive control (634 bp band of gus A gene present in pCAMBIA1381 plasmid), NC negative control (DNA fron nontransformed plant), lanes 1–11 putative transformants

Fig. 4 Southern blot hybridization of T0 putative transformants in Eleusine coracana. M molecular marker, PC positive control (PCR amplified hptII gene), NC negative control (DNA from nontransformed plants), lanes 1–11 putative transformants

Fig. 5 Selectable marker (hptII) gene amplification in T1 generation plants of Eleusine coracana. M molecular marker, PC positive control (684 bp band of hptII gene present in pCAMBIA-1381 plasmid), NC negative control (DNA from nontransformed plant), lanes 1–10 transgenic plants

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finger millet. The efficiency of transformation obtained was 45.3% and higher than those reported earlier for finger millet transformation (Latha et al. 2005). The helium pressure had a significant effect on microprojectile bombardment mediated genetic transformation. Low pressures could be correlated to the reduced transient GUS expression, as microcarriers were not able to reach recipient tissue. On the other hand, higher pressures caused injury of the cells due to increased penetration force. We found that helium pressure of 1,100 psi gave highest transient GUS expression and efficiency of transformation in Eleusine. Similarily, a higher level of transient GUS expression has been reported in banana (Sreeramanan et al. 2005), maize (Petrillo et al. 2008), sugarcane (Kim et al. 2011) and rice (Anoop and Gupta 2004) using 1,100 psi rupture disk pressure, whereas, maximum GUS expression was reported in sorghum when immature embryos were bombarded at 1,300 psi rupture disk pressure and 6 cm target distance (Tadesse et al. 2003). The gap of 3 cm between the rupture disk and macrocarrier was an important factor in improving stable transformation. While in previous investigations on wheat 6 cm distance from rupture disk to macrocarrier was reported to be optimum (Delporte et al. 2005). The distance from the macrocarrier to target tissue can also affect the velocity of microparticles and consequently transformation rates (Petrillo et al. 2008). This distance should be optimized to provide even distribution of DNA-coated microcarrier over the target tissue without damaging it (Tadesse et al. 2003). Tissue dislocation and mechanical damage was observed at too short microprojectile travel distance. As the distance increased the particle velocity and depth of their penetration decreased so that a lower number of cells could receive DNA. We found significantly higher transient GUS expression and transformation efficiency when calli were placed 12 cm away from macrocarrier. Contrary to this, 9 cm microprojectile travel distance was reported to be optimum for rice calli (Ramesh and Gupta 2005), wheat (Gharanjik et al. 2008) and cumin embroys (Singh et al. 2010). Two different types of microparticles (gold/tungsten) were compared for their efficiency in finger millet transformation. Gold particles are round, homogenous in sizes and shapes then tungsten particles thus they are often preferred for particle bombardment. They are biologically inert, non-toxic and do not degrade DNA bonds. On the other hand, tungsten particles are highly heterogeneous in size and shape, toxic and can also acidify solutions and catalyse plasmid DNA degradation (Sanford et al. 1993). Although Latha et al. (2005) reported the successful application of tungsten particles in finger millet transformation, however, we found that use of gold particles for gene delivery resulted into better efficiency of transformation in E. coracana. Depending upon genotype and

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species, size of gold particles was also an important factor that govern the transformation efficiency of monocots (Sood et al. 2011). A very small microcarrier will have a lower penetration force and a bigger one will increase tissue damages (Klein et al. 1988). In our study, we found that 1.0 lm size of microcarrier gave maximum transient GUS expression and efficiency of transformation in finger millet than smaller or larger size of gold particles. Similarly, Albert et al. (2010) used 1.0 lm gold microparticles for Cymbidium transformation. Contrary to this, Takumi et al. (1994) have found that 1.6 lm gold particle was better than 1.0 lm for transformation of einkorn wheat. Whereas, Reggiardo et al. (1991) reported that microprojectile size was not important for the transformation of maize coleoptiles. Double bombardment (rotating the plate by 908) results in better coverage of the target area and increases the efficiency of transformation. Whereas, triple bombardment can cause higher tissue damage particularly with higher helium pressures. In the present study double bombardment per Petri plate was found to be optimum in terms of transient GUS expression and efficiency of transformation. Our results are in line with previous results where double bombardment has been shown to increase the efficiency of transient GUS expression in banana (Sreeramanan et al. 2005) and Brazilian maize inbred lines (Petrillo et al. 2008). But Rasco-Gaunt et al. (1999) reported no significant difference in GUS expression while carrying out single and multiple bombardments on wheat tissues. The penetration of microparticles destructed intracellular lipid membrane structure and caused ethylene accumulation (Imaseki 1986). Osmotic treatment cause plasmolysis of tissue that generally maintains the pressure potential of wounded cells and thus prevent the cell damage and leakage of protoplasm (Ye et al. 1994). In our study sorbitol was found better than mannitol and sucrose for transient GUS expression and transformation efficiency. Whereas, addition of mannitol and sorbitol in the medium for osmotic treatment of maize suspension cells (Vain et al. 1993) and rice calli (Cho et al. 2004) increased the rate of transient and stable transformation. The effect of different concentrations of sorbitol was also tested on transient GUS expression and transformation efficiency. We found that 0.4 M concentration of sorbitol gave highest percent GUS expression and efficiency of transformation. On the contrary, Vasil et al. (1992) observed that in wheat calli, 0.25 M mannitol increased transient GUS expression several fold but at 0.5 M, expression was reduced. Vain et al. (1993) found that treatment of embryogenic maize suspension cultures with 0.2 M sorbitol combined with 0.2 M mannitol 4 h prior to and 16 h after bombardment, gave 2.7 fold increase in transient GUS expression, with 6.8 fold increase in recovery of stably transformed clones.

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In conclusion we have established a simple and efficient microprojectile mediated genetic transformation protocol in finger millet. Our results exhibit the possibility of stable transformation of finger millet through direct gene transfer. The optimized protocol can be applied to produce transgenic finger millet with improved agronomic traits. Acknowledgments We thank UGC, New Delhi for providing Senior Research Fellowship to Swati Jagga–Chugh, UGC, New Delhi for providing postdoctoral fellowship to Dr. Manju Sharma and CSIR, New Delhi for providing Senior Research Fellowship to Aditi Kothari–Chajer.

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