efficient genetic transformation of jatropha curcas l. by microprojectile bombardment using embryo...

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Industrial Crops and Products 33 (2011) 67–77 Contents lists available at ScienceDirect Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes Mukul Joshi, Avinash Mishra , Bhavanath Jha Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), G. B. Marg, Bhavnagar 364021, Gujarat, India article info Article history: Received 22 April 2010 Received in revised form 5 August 2010 Accepted 3 September 2010 Keywords: Biolistic Jatropha Regeneration Southern Transformation Transgenic abstract An efficient and reproducible protocol was established for genetic transformation in Jatropha curcas through microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-cultured for 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physical parameters. The frequency of transient gus expression and survival of putative transformants were taken into consideration for the assessment of physical parameters. Statistical analysis reveal that microcarrier size, helium pressure and target distance had significant influence on transformation efficiency. Among different variables evaluated, microcarrier size 1 m, He pressure 1100 and 1350 psi with a target dis- tance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressure and target distance on the frequency of gus expression and survival of putative transformants. Selection of putative transformants was done with increasing concentrations (5–7 mg L 1 ) of hygromycin. The inte- gration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kb bands of hptII and gus gene respectively from the T 0 transgenics and Southern blot analysis using PCR amplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was made with optimized conditions using particle gene gun and establishing a stable transformation in J. curcas with 44.7% transformation efficiency. The procedure described will be very useful for the introgression of desired genes into J. curcas and the molecular analysis of gene function. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Jatropha curcas, a drought-resistant shrub, is widely distributed in the tropical and sub-tropical areas of Central and South Amer- ica, Africa, India and South East Asia (King et al., 2009). Jatropha is associated with land reclaimation, short growth period, easy adaptation to different kinds of marginal and semi-marginal lands. Drought endurance and avoidance by grazing animals have made this plant species quite profitable for cultivation. Jatropha usually grows below 1400 m of elevation from sea level and requires a min- imum rainfall of 250 mm and an optimum rainfall between 900 and 1200 mm (Maes et al., 2009). Abbreviations: BA, 6-benzyladenine; GA3, gibberellic acid; gus, -glucuronidase; hpt, hygromycin-phosphotransferase; IAA, indole-3-acetic acid; MS, Murashige and Skoog basal salt media; TDZ, 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron); X-Gluc, 5-bromo,4-chloro,3-indolyl, -d-glucuronide. Corresponding authors. Tel.: +91 278 2567352; fax: +91 278 2570885. E-mail addresses: [email protected] (A. Mishra), [email protected] (B. Jha). Recently, attention has been drawn to the high oil content (50–60%) of J. curcas seeds that can be easily processed to partially or fully replace petroleum based diesel fuel (Forson et al., 2004; Ilham and Saka, 2010). Jatropha oil contains approximately 15% free FA (fatty acid) and Jatropha oil biodiesel have approximately 80% unsaturated FA (Berchmans and Hirata, 2008). Oleic acid is the dominant FA in Jatropha biodiesel and over 97% conversion to FAME (fatty acid methyl esters) can be achieved for Jatropha oil. Jat- ropha biodiesel contain 45.79% oleic acid (18:1), 32.27% linoleic acid (18:2), 13.37% palmitic acid (16:0) and 5.43% stearic acid (18:0). Palmitic and stearic acid are the major saturated FA found in Jat- ropha biodiesel (Chhetri et al., 2008). Thus, the use of Jatropha for large-scale biodiesel production is of great interest in concern to solve fuel shortage and increasing the income of farmers (Openshaw, 2000; Zhou et al., 2006; King et al., 2009). Jatropha cultivation not only provides biodiesel but also ensures that agricultural land devoted to food crop production will not become a wasteland. Jatropha is well distributed in India (Sunil et al., 2009) that encourages its use as an alternative source to energy security in the country through biofuel production. Hot and humid weather is prerequisite for good germination of seed and growth of Jatropha plants. Jatropha can grow on gravelly 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.09.002

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Page 1: Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

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Industrial Crops and Products 33 (2011) 67–77

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l homepage: www.e lsev ier .com/ locate / indcrop

fficient genetic transformation of Jatropha curcas L. by microprojectileombardment using embryo axes

ukul Joshi, Avinash Mishra ∗, Bhavanath Jha ∗

iscipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR),. B. Marg, Bhavnagar 364021, Gujarat, India

r t i c l e i n f o

rticle history:eceived 22 April 2010eceived in revised form 5 August 2010ccepted 3 September 2010

eywords:iolistic

atrophaegeneration

a b s t r a c t

An efficient and reproducible protocol was established for genetic transformation in Jatropha curcasthrough microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-culturedfor 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physicalparameters. The frequency of transient gus expression and survival of putative transformants were takeninto consideration for the assessment of physical parameters. Statistical analysis reveal that microcarriersize, helium pressure and target distance had significant influence on transformation efficiency. Amongdifferent variables evaluated, microcarrier size 1 �m, He pressure 1100 and 1350 psi with a target dis-tance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressureand target distance on the frequency of gus expression and survival of putative transformants. Selection

−1

outhernransformationransgenic

of putative transformants was done with increasing concentrations (5–7 mg L ) of hygromycin. The inte-gration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kbbands of hptII and gus gene respectively from the T0 transgenics and Southern blot analysis using PCRamplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was madewith optimized conditions using particle gene gun and establishing a stable transformation in J. curcaswith 44.7% transformation efficiency. The procedure described will be very useful for the introgressionof desired genes into J. curcas and the molecular analysis of gene function.

. Introduction

Jatropha curcas, a drought-resistant shrub, is widely distributedn the tropical and sub-tropical areas of Central and South Amer-ca, Africa, India and South East Asia (King et al., 2009). Jatrophas associated with land reclaimation, short growth period, easydaptation to different kinds of marginal and semi-marginal lands.

rought endurance and avoidance by grazing animals have made

his plant species quite profitable for cultivation. Jatropha usuallyrows below 1400 m of elevation from sea level and requires a min-mum rainfall of 250 mm and an optimum rainfall between 900 and200 mm (Maes et al., 2009).

Abbreviations: BA, 6-benzyladenine; GA3, gibberellic acid; gus, �-glucuronidase;pt, hygromycin-phosphotransferase; IAA, indole-3-acetic acid; MS, Murashige andkoog basal salt media; TDZ, 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron);-Gluc, 5-bromo,4-chloro,3-indolyl, �-d-glucuronide.∗ Corresponding authors. Tel.: +91 278 2567352; fax: +91 278 2570885.

E-mail addresses: [email protected] (A. Mishra), [email protected] (B. Jha).

926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2010.09.002

© 2010 Elsevier B.V. All rights reserved.

Recently, attention has been drawn to the high oil content(50–60%) of J. curcas seeds that can be easily processed to partiallyor fully replace petroleum based diesel fuel (Forson et al., 2004;Ilham and Saka, 2010). Jatropha oil contains approximately 15%free FA (fatty acid) and Jatropha oil biodiesel have approximately80% unsaturated FA (Berchmans and Hirata, 2008). Oleic acid isthe dominant FA in Jatropha biodiesel and over 97% conversion toFAME (fatty acid methyl esters) can be achieved for Jatropha oil. Jat-ropha biodiesel contain 45.79% oleic acid (18:1), 32.27% linoleic acid(18:2), 13.37% palmitic acid (16:0) and 5.43% stearic acid (18:0).Palmitic and stearic acid are the major saturated FA found in Jat-ropha biodiesel (Chhetri et al., 2008).

Thus, the use of Jatropha for large-scale biodiesel production isof great interest in concern to solve fuel shortage and increasingthe income of farmers (Openshaw, 2000; Zhou et al., 2006; Kinget al., 2009). Jatropha cultivation not only provides biodiesel butalso ensures that agricultural land devoted to food crop production

will not become a wasteland. Jatropha is well distributed in India(Sunil et al., 2009) that encourages its use as an alternative sourceto energy security in the country through biofuel production.

Hot and humid weather is prerequisite for good germination ofseed and growth of Jatropha plants. Jatropha can grow on gravelly

Page 2: Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

6 ps and

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explants were transferred to shoot regeneration medium (SRM;MS + 3% sucrose + 0.8% agar + 2.22 �M BA + 0.49 �M IBA + 1.45 �M

8 M. Joshi et al. / Industrial Cro

r sandy soils with low nutrient content and can also grow well inrid areas, where irrigation is provided. Jatropha cultivation is stillimited by some abiotic and biotic stresses, especially salt, cold andnsect pest. Extensive salt farming, scanty rainfall and uncontrolledse of ground water for industrial purposes are few reasons of grad-ally increasing salinity and drought. The area under cultivation

s fast getting depleted and becoming unsuitable for agriculturalrops. The barrier for profitable cultivation of Jatropha is abiotictress and it requires urgent steps to develop abiotic tolerant plantshat can cope with adverse conditions involving wasteland useor sustainable development. Similarly, Jatropha is attacked andffected by many biotic stresses, i.e. insect pests, which hamperts growth and productivity (Shanker and Dhyani, 2006) contraryo popular belief that toxicity and insecticidal properties of J. curcasre sufficient deterrent for insects that cause economic damage inlantation.

Therefore, additional genetic tools are required to explore theotential resources of J. curcas and provide additional geneticain. Recent advances in gene manipulations, DNA technology andenetic transformation offer a credible approach for the develop-ent of transgenics, tolerant to abiotic stress, resistant to insects

nd with improved agronomical traits. Moreover, direct geneticransformation has become a method of choice for basic plantesearch as well as a principal technology for generating trans-enic plants. Jatropha improvement requires an efficient geneticransformation and plant regeneration system.

In recent years, plantlet regeneration from different J. curcasxplants, i.e. cotyledon, petiole, hypocotyl, epicotyl, leaf tissues andtem have been successfully obtained (Sujatha and Mukta, 1996;ardana et al., 2000; Wei et al., 2004; Rajore and Batra, 2005;ujatha et al., 2005; Jha et al., 2007; Deore and Johnson, 2008;ingh et al., 2010) and an efficient as well as reproducible plantegeneration procedure has been established. The most widelysed methods of genetic transformation are the direct gene trans-er method using particle gun and the vector-mediated methodsing Agrobacterium. Both these methods have their own advan-ages and limitations (Potrykus, 1991; Sharma et al., 2005; Altpetert al., 2005). Preliminary Agrobacterium mediated genetic trans-ormation has been done using cotyledonary disc which revealusceptibility of Jatropha explants to Agrobacterium mediatedransformation (Li et al., 2006, 2008) and leaf explants (Kumar etl., 2010).

Particle bombardment facilitates a wide range of transforma-ion strategies, high molecular weight DNA delivery into plantells, simultaneous multiple gene transformation with no biologicalonstraints and host limitations (Altpeter et al., 2005). More-ver, diverse cell types can also be targeted efficiently for foreignNA delivery. However, in Agrobacterium mediated transforma-

ion, choice of explants is very limited. Jatropha cotyledons are moreusceptible to Agrobacterium infection than other explants such asetioles, hypocotyls, epicotyls or leaves (Li et al., 2006) and trans-ormation efficiency is also dependent on Agrobacterium strain. Onhe other hand, the widely held belief that Agrobacterium medi-ted transformation is more precise and controllable than particleombardment is beginning to be demystified (Batista et al., 2008).report on bombardment strategies shows the recovery of trans-

enic plants, containing intact, single-copy integration events withigh-level transgene expression, especially in non-model plant sys-ems (Altpeter et al., 2005).

In this investigation, physical parameters for direct gene trans-er using particle gun are optimized using high quality CP-9 cultivarf J. curcas. It is a successful attempt of transformation of Jat-opha plants through direct gene transfer using particle gun and

dequately exhibiting the possibility of stable transformation in Jat-opha. This study has long-term implications in genetic engineeringf J. curcas for desired traits.

Products 33 (2011) 67–77

2. Materials and methods

2.1. Plant material and culture conditions

J. curcas seeds of CP-9 cultivar were used for regeneration andtransformation. Mature decoated seeds of Jatropha were surfacesterilized with 0.1% (w/v) mercuric chloride for 15 min and washed4–6 times with sterile distilled water. Embryo explants were dis-sected out aseptically from endosperm and cotyledons removedcarefully. Explants were pre-cultured for five days on optimizedsolid MS basal media (Murashige and Skoog, 1962) (pH 5.8) sup-plemented with 2.22 �M BA, 0.8% (w/v) agar and 3% (w/v) sucrose.All cultures were maintained under controlled laboratory condi-tions at 25 ± 2 ◦C under a 16/8 h light/dark photoperiod with coolwhite fluorescent lamp of 35 �mol m−2 s−1 light intensity.

2.2. Transformation with particle gun

Explants were arranged aseptically in a circle with diameterof 25 mm on same media just before the bombardment. PlasmidpCAMBIA 1301 was isolated by using plasmid Miniprep kit (Qiagen,Germany) following manufacturer’s protocol. Transformation con-ditions were determined using the plasmid pCAMBIA 1301, whichharbours the gus reporter gene and the selectable hptII gene, bothcontrolled by the cauliflower mosaic virus (CaMV) 35S promoter.

2.2.1. Preparation of microcarriersMicrocarriers (0.5 mg gold) coated with 1 �g of plasmid DNA

and suspended in 50 �l absolute ethanol, were used as a stan-dard for each bombardment. Gold microparticles were suspendedin 1 ml 70% ethanol (v/v) by vigorous vortexing for 3–5 min fol-lowed by soaking for 15 min. Microparticles were washed 3 timeswith 1 ml sterile water by spinning for 30 s in a microfuge. Afterthird wash, microparticles were suspended in sterile 50% glyceroland coated with plasmid DNA (pCAMBIA 1301) using CaCl2 (2.5 M)and spermidine (0.1 M) precipitation method. After 10 min incuba-tion on ice, the supernatant was removed and pellet was washedwith 70% (v/v) ethanol followed by washing with absolute ethanol.After washing, the particle DNA pellet was re-suspended in abso-lute ethanol for bombardments. Care was taken to ensure uniformparticle distribution and minimize agglomeration.

2.2.2. Microprojectile bombardmentBombardments were done with biolistic gene gun (PDS

1000/He, Bio-Rad) under a vacuum of 27 in. of Hg, a 25 mm dis-tance from rupture disc to macrocarrier and a 10 mm macrocarrierflight distance for all bombardments. The variables to be opti-mized included five rupture disc pressures (650, 900, 1100, 1350and 1550 psi), four microprojectile travel distances (3, 6, 9, and12 cm) and microcarrier size (gold particle size 0.6, 1.0 and 1.6 �m).Non-bombarded embryo axes and embryo axes bombarded withuncoated microcarriers were used as controls.

2.3. Selection and regeneration of transformants

After bombardment, explants were kept in dark at 25 ◦C for24 h and then transferred to shoot induction medium (SIM), i.e.MS medium containing 3% sucrose, 0.8% agar and plant growthregulators 2.22 �M BA + 2.27 �M TDZ + 0.49 �M IBA. After 15 daysthe explants were transferred to selection medium (same asabove) containing 5 mg L−1 hygromycin. For effective selection, the

GA3) with increasing concentration (6 and 7 mg L−1) of hygromycin.After three cycles of selection, putative transformed shoots weretransferred for approximately 40–60 days to shoot elongation

Page 3: Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

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medium (SEM; MS + 3% sucrose, 0.8% agar + 2.22 �M BA) for furthershoot elongation.

For rooting, elongated shoots were transferred to root inductionmedium (RIM) containing half-strength MS basal salts, 1.47 �M IBAand 2% sucrose. After 4 weeks, plantlets with rooted shoots weretransplanted into pots (5 × 10 cm) covered with transparent plasticlids and maintained under high humidity for 7–10 days, thereaftergradually exposed to culture room conditions followed by greenhouse conditions. Established plantlets were transferred to pots forhardening.

2.4. Histochemical GUS assay

Transient gus expression was assessed after 24 h of bombard-ment and randomly 20 transformed embryos per shot per platewere selected (Jefferson et al., 1987). Whole plantlets and leavesof transgenic lines (4–5-month-old after bombardment) wereassayed for the constitutive expression of gus gene. GUS assay wasdone by incubating the tissues in freshly prepared GUS assay buffer(1 g L−1 X-Gluc with 0.05 M Na2HPO4, 0.5 mM K3Fe(CN)6, 0.5 mMK4Fe(CN)6, 10 mM EDTA and 0.1% (v/v) Triton X-100) for 12 h at37 ◦C. Thereafter tissues were destained with 70% alcohol to exam-ine and count blue spots. Explants with at least one discrete blueregion on the tissue were scored for GUS positive.

2.5. Molecular analysis

2.5.1. PCR amplificationThe genomic DNA of transformants was isolated using CTAB

method (Doyle and Doyle, 1987). Transformation was confirmedby PCR with gus (reporter gene) specific primers (F: 5′-GAT CGCGAA AAC TGT GGA AT-3′ and R: 5′-TGA GCG TCG CAG AAC ATT AC-3′) and hptII (hygromycin selection marker gene) specific primers(F: 5′-TTC TTT GCC CTC GGA CGA GTG-3′ and R: 5′-ACA GCG TCT CCGACC TGA TG-3′) using initial denaturation temperature of 94 ◦C for10 min, subsequent 35 cycles of 94 ◦C denaturation for 1 min, 60 ◦Cannealing for 1 min, 72 ◦C extension for 1.5 min and final extensionat 72 ◦C for 7 min.

2.5.2. Southern hybridizationGenomic DNA (20 �g) from transformants was digested with

EcoRI, separated by electrophoresis in a 0.8% agarose gel and trans-ferred onto a Hybond N+ membrane (Amersham Pharmacia, UK)by capillary method using alkaline transfer buffer (0.4N NaOH with1 M NaCl). The membrane was air-dried and DNA was fixed to themembrane by UV cross-linking using 56 mJ cm−2 energy for 1 minin a UVC 500 cross-linker (Amersham Biosciences, UK). Blot washybridized with PCR-generated probe for hptII gene labeled withDIG-11-dUTP, amplified from plasmid pCAMBIA 1301 using 0.1 mMDIG-11-dUTP, 1.9 mM dTTP and Taq DNA polymerase, followingmanufacturer’s user guide (Roche, Germany). Purified pCAMBIA1301 and PCR amplified hptII gene served as a positive controlwhile DNA from non-transformed plant as a negative control. Pre-hybridization and hybridization were carried out at 68 ◦C overnightin DIG EasyHyb buffer solution (Roche, Germany). The membranewas then washed 2–3 times at room temperature for 5 min in2× SSC, 0.1% SDS, and twice for 15 min in 0.2× SSC, 0.1% SDSat 68 ◦C. The hybridized membrane was detected by using CDP-Star chemiluminescent as substrate, following manufacturer’s userguide (Roche, Germany) and signals were visualized on X-ray filmafter 30 min.

2.6. Statistical analysis

Each treatment consisted of at least two plates and was repli-cated thrice. Frequency of GUS activity was calculated as number

Page 4: Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

70 M. Joshi et al. / Industrial Crops and Products 33 (2011) 67–77

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ig. 1. Effect of microcarrier size and helium pressure on (a) transient gus expressapital case and lower letters are significantly different for their respective dataolynomial regression (order value 2) for grand mean value.

f embryos showing gus expression to the total number of explantstained after bombardment and is expressed as percentage. Data onhe number of embryo axes showing transient gus expression wasubjected to analysis of variance (ANOVA) for analysis to deter-ine differences (Sokal and Rohlf, 1995) and were expressed asean ± SE. A Tukey’s HSD multiple comparison of mean test was

sed when significant differences were found and P < 0.01 was con-idered as significant.

. Results

Decotyledonated embryos were elongated up to 0.5 cm withistinct proliferated meristematic regions within 5 days of incu-ation on medium (MS basal of pH 5.8 + 0.8% (w/v) agar + 3% (w/v)ucrose + 2.22 �M BA). The elongated embryos were excised andicroprojectile bombardment mediated transformation was opti-ized on these embryonic meristematic tissues. For assessment of

he effect of microcarrier size, helium pressure and microprojectileravel distance during bombardment, frequency of transient gusxpression and survival of putative transformants after third cyclef selection were taken into consideration.

.1. Microcarrier size vs. helium pressure

Frequency of transient gus expression was 43.9 ± 7.3, 59.1 ± 5.7nd 66.1 ± 6.6, however, frequency of shoot survival after threeounds of hygromycin selection was 13.2 ± 2.16, 16.14 ± 2.95 and.95 ± 0.7 for 0.6, 1.0 and 1.6 �m gold microcarriers (Table 1nd S1). Transient gus expression was significantly increased

d (b) survival of shoots after 3rd round of selection. Means ± SE followed by samecording to Tukey’s HSD at P < 0.01 and P < 0.05 respectively. Trend line indicates

(P < 0.05) while frequency of shoot survival decreased (P < 0.01)with increase in microcarrier size. Overall, optimum microcarriersize was observed 1.0 �m while comparing transient gus expres-sion and frequency of shoot survival (S1). Like microcarrier size,transient gus expression was increased while frequency of shootsurvival decreased concomitantly with helium pressure (Table 1and S2). Maximum transient gus expression was observed at 1100and 1350 psi for 0.6 and 1.0 �m microcarrier. Apart to this, sig-nificant transient gus expression was also observed at 1550 psifor 1.6 �m microcarrier (Fig. 1a). Optimum transient gus expres-sion was observed at 1100 and 1350 psi He pressure (Table 1 andFig. 1a). Maximum survival of shoots was observed at 650 and900 psi He pressure for 0.6 and 1.6 �m while 1100 psi for 1.0 �mmicrocarriers (Table 2 and Fig. 1b). Overall optimum He pres-sure was observed 1100 and 1350 psi and microcarrier size 1.0 �mindependent to microprojectile travel distance while comparingtransient gus expression and frequency of shoot survival (Fig. 1aand b).

3.2. Microcarrier size vs. microprojectile travel distance

The mean transient gus expression was 64.97 ± 4.84,68.6 ± 8.521, 51.15 ± 5.84 and 40.88 ± 8.37 for target distances of3, 6, 9, and 12 cm respectively (Table 1, Fig. 2a and S3). Frequency

of shoot survival increased concomitantly with target distance(Table 2, Fig. 2b and S3) while comparing transient gus expressionand frequency of shoot survival (Fig. 2a and b), optimum traveldistance was observed 9 and 12 cm and microcarrier size 1.0 �mindependent to He pressure.
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.3. Helium pressure vs. microprojectile travel distance

The mean transient gus expression was 53.18 ± 12.11,7.63 ± 9.1, 69.7 ± 15.09, 73.9 ± 7.68 and 57.5 ± 6.19 for 650,00, 1100, 1350 and 1550 psi He pressure respectively (Table 1

able 2ffect of microcarrier size, helium pressure and target distance on transformation efficien

Gold particle size Helium pressure(psi)

Microprojectile traveldistance (cm)

No. of explantbombarded

0.6 �m 650 3 1206 1279 133

12 123900 3 155

6 1439 172

12 1491100 3 152

6 1419 125

12 1561350 3 131

6 1299 140

12 1421550 3 121

6 1269 136

12 139

1.0 �m 650 3 1926 1889 187

12 195900 3 168

6 1779 175

12 1641100 3 173

6 1569 188

12 1811350 3 178

6 1939 192

12 1741550 3 164

6 1759 192

12 190

1.6 �m 650 3 1786 1979 163

12 184900 3 187

6 1939 187

12 1751100 3 173

6 1859 181

12 1781350 3 199

6 1929 158

12 1631550 3 144

6 1479 132

12 108

ubculture was done at 3weeks interval onto respective medium containing 5, 6 and 7hown in bold numerical.

Products 33 (2011) 67–77 71

and Fig. 3a). Frequency of shoot survival declined with increasein target distance (Table 2 and Fig. 3b) while comparing transientgus expression and frequency of shoot survival, optimum traveldistance was observed 9 and 12 cm with He pressure 1100 and1350 psi respectively independent to microcarrier size.

cy.

s Relative frequency oftransient gus expression(percentile)

Frequency of shoot survival (%)

Selection I Selection II Selection III

35.69 92.5 42.5 24.233.43 98.4 50.4 24.431.48 97.7 55.6 20.310.0 91.1 50.4 21.136.9 82.6 41.3 21.334.49 95.1 40.6 20.332.68 87.8 53.5 22.730.87 91.9 53.7 26.838.10 75.0 28.9 19.134.04 79.4 34.8 16.336.6 96.8 37.6 13.632.08 98.1 41.0 15.440.60 71.8 18.3 2.338.71 95.3 14.7 1.636.0 87.1 29.3 3.632.98 93.0 26.8 1.452.71 70.2 4.1 051.66 75.4 5.6 033.58 72.1 19.1 3.750.0 73.4 19.4 5.8

50.90 89.1 40.1 23.452.11 91.5 44.1 25.048.04 92.5 48.7 19.814.76 95.9 45.1 27.238.25 83.3 48.2 12.535.30 88.7 52.0 11.335.09 95.4 46.3 13.732.08 97.6 53.7 12.842.92 89.6 35.3 10.440.06 91.0 32.1 21.268.07 98.4 58.0 44.735.54 99.4 47.5 32.047.29 86.5 19.7 041.42 91.2 17.1 8.336.30 89.1 31.8 19.864.00 97.7 49.4 39.158.28 71.3 2.4 054.97 76.0 8.0 036.75 72.4 1.6 035.39 77.4 2.1 1.6

71.08 80.9 32.0 068.68 78.7 39.1 1.564.61 92.6 33.7 4.359.64 93.5 32.1 11.473.34 86.1 11.2 056.18 87.0 14.5 5.750.75 88.8 13.4 6.444.88 90.9 10.3 5.776.96 71.1 1.7 052.11 81.6 1.6 057.38 91.7 8.3 1.745.78 88.8 6.7 2.264.06 70.9 2.0 061.90 72.9 3.1 055.27 78.5 4.4 048.95 82.8 5.5 0

100 67.4 0 079.82 68.7 0 062.05 74.2 0 053.62. 84.3 0 0

mg hygromycin/l for selections I, II and III, respectively. Optimized conditions are

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72 M. Joshi et al. / Industrial Crops and Products 33 (2011) 67–77

y = -3.477x2 + 8.414x + 61.44R² = 0.918

80

90

100

(%)

0.6 μm Gold

1.0 μm Gold

1.6 μm Gold

Mean

P l (M )

bde

30

40

50

60

70

ent g

usex

pres

sion

Poly. (Mean)a

b

c

e

0

10

20

30

3 cm 6 cm 9 cm 12 cm

Tran

si acd

Microprojec�le travel distance (cm)

a

y = 0.097x2 + 1.555x + 5.807R² 0 992

35 0.6 μm Gold1.0 μm Gold R² = 0.992

20

25

30

h oot

s su

rviv

al (%

)

μ1.6 μm GoldMeanPoly. (Mean)

cg

dh

eij

0

5

10

15

Freq

uenc

y of

Sh a

abcde

bf

fghi

dh

j

3 cm 6 cm 9 cm 12 cm

Microprojec�le travel distance (cm)

b

Fig. 2. Effect of microcarrier size and target distance on (a) transient gus expression (b) survival of shoots after 3rd round of selection. Means ± SE followed by similar lettersare significantly different according to Tukey’s HSD at P < 0.05. Trend line indicates polynomial regression (order value 2) for grand mean value.

Fig. 3. Effect of helium pressure and target distance on (a) transient gus expression (b) survival of shoots after 3rd round of selection. Means ± SE followed by similar lettersare significantly different according to Tukey’s HSD at P < 0.01. Trend line indicates polynomial regression (order value 2) for grand mean value.

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Fr(ss

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.4. Tissue culture and histochemical GUS assay

Transformed embryos per shot per plate were selected ran-omly after 24 h of bombardment and transient gus expression

ig. 4. Microprojectile bombardment-mediated transformation, selection, regeneration aadicle part of embryo and (d) inner side (transverse section) of embryo axis after 24 h of ba). Selection of transformants (e) 1st round (28 days old), (f) 2nd round (48 days old) anhoot regeneration (90 days old), (i) shoot elongation (140 days old), (j) rooting (170 dahown in insight is 200 days old). GUS assay of (l) non-transformed leaf, (m) whole plant

Products 33 (2011) 67–77 73

was assessed for each combination of parameters (Fig. 4a–d). Puta-tive transformants were transferred to shoot induction medium(SIM; MS medium + 3% sucrose + 0.8% agar + 2.22 �M BA + 2.27 �MTDZ + 0.49 �M IBA) after 24 h of bombardment for 15 days.

nd GUS assay. Transient gus expression on (a) embryo axis, (b) whole embryo, (c)ombardment. Five days elongated embryo ready to transform was shown as insightd (g) 3rd round (68 days old) on hygromycin. Regeneration of transformants; (h)

ys old) and (k) hardening of transgenic plant (3-month-old after hardening; plantlet and (n) leaf of transgenic plant.

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7 ps and Products 33 (2011) 67–77

Tc2mIoa7Dsrig3TpeftBtathat

3

pbrgayggs(td

4

awei(ie(tmcampb2tbt

Fig. 5. Molecular analysis of transgenic plants. (a) PCR amplification of the hptIIgene (963 bp), (b) gus gene (1286 bp) and (c) southern analysis of randomly selectedputative transgenic plants transformed with optimized conditions. Lane ML: molec-ular weight marker ladder; lane �M: �DNA EcoR1/HindIII double digest molecular

4 M. Joshi et al. / Industrial Cro

he transformants were transferred to selection medium (SIM)ontaining 5 mg L−1 hygromycin for 1st round of selection for1 days (Fig. 4e), thereafter transferred to shoot regenerationedium (SRM; MS + 3% sucrose + 0.8% agar + 2.22 �M BA + 0.49 �M

BA + 1.45 �M GA3) containing 6 mg L−1 hygromycin for 2nd roundf selection for 21 days (Fig. 4f). For effective selection, regener-ted transformants were transferred to same medium containingmg L−1 hygromycin for 21 days for 3rd round of selection (Fig. 4g).uring selection on medium with hygromycin, non-transgenic tis-

ues gradually turned brown, while putative transformed sectorsemained green and exhibited slow growth (Fig. 4e–g). The max-mum transformation efficiency 44.7% was observed with 1 �Mold, 1100 psi He pressure and 9 cm travel distance followed by9.1% with 1350 psi He pressure and 12 cm travel distance (Table 2).ransformation efficiency is calculated as number of PCR positivelants survived after third round of selection with respect to totalmbryos bombarded. After three cycles of selection, putative trans-ormed shoots were transferred for approximately 40–60 days tohe MS medium containing 3% sucrose, 0.8% agar and 2.22 �MA for further shoot elongation (Fig. 4h and i) and subsequentlyransferred to root induction medium (1/2MS + 2% sucrose + 0.8%gar + 1.47 �M IBA) for approximately 28 days (Fig. 4j). Putativeransgenics with rooted shoots were transplanted into pots forardening (Fig. 4k). Meanwhile whole plantlets and leaves weressayed for the constitutive expression of gus gene using non-ransformed leaf as control (Fig. 4l–n).

.5. Molecular analysis of transgenic plants

The confirmation of genetic transformation was based on theresence of reporter gene gus and selectable marker gene hptIIy PCR amplification of expected bands of sizes 1.28 and 0.96 kb,espectively (Fig. 5a and b). Randomly 3-month-old putative trans-enic plants transformed with optimized conditions and survivedfter three rounds of selection were selected for the molecular anal-sis. All plants tested were observed positive for both gus and hptIIenes. Southern analysis was done following PCR confirmation ofus and hptII genes for the same transformants. Southern analy-is revealed detectable signals in PCR positive plants for hptII geneFig. 5c). Generally, transformants showed single copy while tworansgenic plants contained multiple (2 and 3) copies of the intro-uced gene.

. Discussion

Gene introgression by particle bombardment is most efficientnd consistent, genotype independent versatile physical processith no biological constraint (Altpeter et al., 2005). A simple and

fficient microprojectile mediated genetic transformation methodn Jatropha was established with 44.7% transformation efficiencyFig. 6). Different physical parameters need to be carefully exam-ned in particle bombardment that could enhance transient GUSxpression and lead to stable integration of the introduced genesSailaja et al., 2008). In this study, different physical parame-ers such as microcarrier size, velocity of particle delivery and

icroprojectile travel distance were optimized individually and in-ombination considering the frequency of transient gus expressionnd survival of putative transformants. Microprojectile bombard-ent with embryonic axis as target tissues has been used for the

roduction of successful transgenic lines in many plants, i.e. soy-

ean (McCabe et al., 1988; Rech et al., 2008), cowpea (Ivo et al.,008), peanut (Brar et al., 1994; Livingstone and Birch, 1999), cas-or (Sailaja et al., 2008) and corn (Lowe et al., 2009). Microprojectileombardment is independent to any kind of target tissue but inhis study embryo axes, pre-cultured for 5 days on MS medium

weight marker; lane PC1: PCR amplified hptII gene (positive control); lane PC2: PCRamplified gus gene (positive control); lane PC3: pCAMBIA 1301 (positive control);lane NTC: non-transformed plantlet (negative control), and lanes J1–J8: putativetransgenic plants.

were taken as both castor and Jatropha belong to Euphorbiaceaefamily and embryo axis was found suitable for stable genetic trans-formation in castor (Sailaja et al., 2008) and regeneration (Sujathaand Mukta, 1996). Embryo was pre-cultured for 5 days to maxi-mize the probability of stable transformation as actively dividingcells have ability to survive and grow under stress imposed duringbombardment process (Sailaja et al., 2008).

Frequency of transient gus expression was significantlyincreased (P < 0.05) and frequency of shoot survival decreased

(P < 0.01) with an increase in particle size. Similarly, transientgus expression was increased while frequency of shoot survivaldecreased concomitantly with helium pressure. High pressure andlarge microcarriers penetrated into deep cell layers and integratedwith genome, hence more transient gus expression was observed.
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M. Joshi et al. / Industrial Crops and Products 33 (2011) 67–77 75

Decoated mature seedsSterilization with 0.1% HgCl2 for 15 min

Embryo axes excised and cultured on MS medium with 2.22 μM BA5 d di l d5 days, radicles removed

Bombarded with gold particles coated with vector(1 μm gold particle size, 1100 psi He pressure and 9 cm microprojectile travel distance)

Incubation in dark for 24 hours(Transient GUS assay)

Transfer to shoot induction medium (SIM)(2.22 μM BA + 2.27 μM TDZ + 0.49 μM IBA)

15 days

Transfer to selection medium (SIM) containing 5 mgL-1 Hygromycin21 days, 1st selection

Shoot regeneration on selection medium (2.22 μM BA + 0.49 μM IBA + 1.45 μM GA3 and 6 mgL-1 hygromycin)

21 days, 2nd selection

Subculture to selection medium(2.22 μM BA + 0.49 μM IBA + 1.45 μM GA3 and 7 mgL-1 hygromycin for shoot regeneration)

21 days, 3rd selection21 days, 3 selection

Subculture of regenerated shoots to shoot elongation medium (SEM) containing 2.22 μM BA28 days

Subculture to SEM for further elongation of shoots28 days

Transfer to root induction medium containing ½MS and 1.47 μM IBA28-35 days

Rooted plants transplanted and acclimatized in plastic pots28-35 days

T f t t i fi ld ditiTransfer to pots in field conditions

F bardma

Sa1megi

tpipoetrfHegsftit

ig. 6. Schematic representation of the complete protocol for microprojectile bomxes.

imultaneously, it also imposed injury leading to decrease in prob-bility of shoot survival. The optimum He pressure was observed100 and 1350 psi and microcarrier size 1.0 �m independent toicroprojectile travel distance, while comparing transient gus

xpression and frequency of shoot survival. In this combination,ene introgression was efficient for transient gus expression lead-ng to maximum shoot survival.

Decline in transient gus expression with increase of travel dis-ance in combination with microcarrier size independent to Heressure could be due to deceleration of the microprojectile veloc-

ty. However, opposite result was observed in combination with Heressure independent to microcarrier size because of accelerationf microcarrier with high pressure. While comparing transient gusxpression and frequency of shoot survival, optimum travel dis-ance was observed 9 and 12 cm for He pressure 1100 and 1350 psiespectively, independent to microcarrier size. The genetic trans-ormation efficiency was determined at different microcarrier size,e pressure and target distance both in terms of transient gusxpression and shoot survival. The study revealed that transientus expression is not the key to analyze transformation efficiency. In

pite of a very high frequency of transient gus expression at 1550 psior 1.6 �m microcarrier, the frequency of surviving explants dras-ically declined during selections because high helium pressurencreased particle acceleration and subsequent target tissue pene-ration leads to injury by DNA coated microcarriers.

ent mediated genetic transformation and regeneration of J. curcas using embryo

Overall optimum parameters were observed for microcarriersize 1.0 �m, He pressure 1100 and 1350 psi with target distance9 and 12 cm respectively by comparing transient gus expressionand frequency of shoot survival. The efficiency of transformationobtained through particle gun gene transfer in the present study is44.7% and higher than those reported earlier for Jatropha (Li et al.,2008; Kumar et al., 2010; Purkayastha et al., 2010). The transforma-tion efficiencies through Agrobacterium-mediated transformationwere 13% with cotyledon disc (Li et al., 2008) and 29% with leafexplants (Kumar et al., 2010), however, Purkayastha et al. (2010)transformed Jatropha using shoot apices by particle bombardmentbut transformation efficiency was not reported. Higher efficiency oftransformation through particle gun bombardment could probablybe due to the higher number of explants tried with varying individ-ual and in-combination physical parameters. High transformationefficiency over previous methods makes optimized protocol (Fig. 6)efficient and thus has the potential to facilitate the genetic mod-ification for trait improvement. The optimized parameters wereefficiently used previously for evergreen coniferous tree Norwayspruce (Walter et al., 1999), maize (Bohorova et al., 1999), rice

(Cho et al., 2004), potato (Ercolano et al., 2004), St. John’s wort(Franklin et al., 2007), Madagascar periwinkle (Guirimand et al.,2009) and Jatropha (Purkayastha et al., 2010). Callus browningis a typical feature of callus cultures derived from the hypocotylof J. curcas (He et al., 2009) and during selection on hygromycin
Page 10: Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

7 ps and

mpgg

lttgt(ct

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hgtittetaot

tfimtfi

A

P

A

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B

B

B

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C

6 M. Joshi et al. / Industrial Cro

edium, non-transgenic tissues gradually turned brown, whileutative transformed sectors remained green and showed slowrowth. Brown tissues result in decreased regenerative ability, poorrowth and subsequent death.

Histochemical GUS assay after the final selection of transformedines, showing constitutive expression of gus gene, confirmedhe efficient integration of gene which is also evident withhe PCR amplification of both gus and selectable marker hptIIene. Generally, in microprojectile bombardment mediated geneticransformation, multiple copies of inserted genes are reportedAltpeter et al., 2005), however, in this work, mostly (75%) singleopy of insertion was observed by southern analysis, confirminghe efficacy of the present method.

. Conclusion

ANOVA reveals highly significant effects of microcarrier size,elium pressure, target distance and their interaction on transientus expression and frequency of transformants survival. Despitehe increase in frequency of transient gus expression with increasen helium pressure and microcarrier size, there was a reduction inhe frequency of surviving shoots and shoots failed to survive afterhird cycle of selection in some combinations of parameters. Theffect of helium pressure and its interaction with target distance onhe frequency of shoot survival was highly significant. Significantnd polygonal correlations of helium pressure and target distancen the frequency of gus expression and shoot survival of putativeransformants were found.

An efficient microprojectile bombardment mediated geneticransformation and plant regeneration protocol was establishedor J. curcas using embryo axes (Fig. 6). The results of the presentnvestigation adequately exhibit the possibility of stable transfor-

ation of Jatropha through direct gene transfer method with 44.7%ransformation efficiency. The optimized protocol will be very use-ul for introduction of desired gene in biofuel plants for the traitmprovement in future.

cknowledgement

The financial support received from CSIR, New Delhi (Networkroject NWP-20) is thankfully acknowledged.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.indcrop.2010.09.002.

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