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

Industrial Crops and Products 33 (2011) 67–77

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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

a r t i c l e

i n f o

Article history: Received 22 April 2010 Received in revised form 5 August 2010 Accepted 3 September 2010

Keywords: Biolistic Jatropha Regeneration Southern Transformation Transgenic

a b s t r a c t 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 distance 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 integration 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 T0 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 America, 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 minimum 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). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.09.002

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. Jatropha 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 Jatropha 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

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or sandy soils with low nutrient content and can also grow well in arid areas, where irrigation is provided. Jatropha cultivation is still limited by some abiotic and biotic stresses, especially salt, cold and insect pest. Extensive salt farming, scanty rainfall and uncontrolled use of ground water for industrial purposes are few reasons of gradually increasing salinity and drought. The area under cultivation is fast getting depleted and becoming unsuitable for agricultural crops. The barrier for profitable cultivation of Jatropha is abiotic stress and it requires urgent steps to develop abiotic tolerant plants that can cope with adverse conditions involving wasteland use for sustainable development. Similarly, Jatropha is attacked and affected by many biotic stresses, i.e. insect pests, which hamper its growth and productivity (Shanker and Dhyani, 2006) contrary to popular belief that toxicity and insecticidal properties of J. curcas are sufficient deterrent for insects that cause economic damage in plantation. Therefore, additional genetic tools are required to explore the potential resources of J. curcas and provide additional genetic gain. Recent advances in gene manipulations, DNA technology and genetic transformation offer a credible approach for the development of transgenics, tolerant to abiotic stress, resistant to insects and with improved agronomical traits. Moreover, direct genetic transformation has become a method of choice for basic plant research as well as a principal technology for generating transgenic plants. Jatropha improvement requires an efficient genetic transformation and plant regeneration system. In recent years, plantlet regeneration from different J. curcas explants, i.e. cotyledon, petiole, hypocotyl, epicotyl, leaf tissues and stem have been successfully obtained (Sujatha and Mukta, 1996; Sardana et al., 2000; Wei et al., 2004; Rajore and Batra, 2005; Sujatha et al., 2005; Jha et al., 2007; Deore and Johnson, 2008; Singh et al., 2010) and an efficient as well as reproducible plant regeneration procedure has been established. The most widely used methods of genetic transformation are the direct gene transfer method using particle gun and the vector-mediated method using Agrobacterium. Both these methods have their own advantages and limitations (Potrykus, 1991; Sharma et al., 2005; Altpeter et al., 2005). Preliminary Agrobacterium mediated genetic transformation has been done using cotyledonary disc which reveal susceptibility of Jatropha explants to Agrobacterium mediated transformation (Li et al., 2006, 2008) and leaf explants (Kumar et al., 2010). Particle bombardment facilitates a wide range of transformation strategies, high molecular weight DNA delivery into plant cells, simultaneous multiple gene transformation with no biological constraints and host limitations (Altpeter et al., 2005). Moreover, diverse cell types can also be targeted efficiently for foreign DNA delivery. However, in Agrobacterium mediated transformation, choice of explants is very limited. Jatropha cotyledons are more susceptible to Agrobacterium infection than other explants such as petioles, hypocotyls, epicotyls or leaves (Li et al., 2006) and transformation efficiency is also dependent on Agrobacterium strain. On the other hand, the widely held belief that Agrobacterium mediated transformation is more precise and controllable than particle bombardment is beginning to be demystified (Batista et al., 2008). A report on bombardment strategies shows the recovery of transgenic plants, containing intact, single-copy integration events with high-level transgene expression, especially in non-model plant systems (Altpeter et al., 2005). In this investigation, physical parameters for direct gene transfer using particle gun are optimized using high quality CP-9 cultivar of J. curcas. It is a successful attempt of transformation of Jatropha plants through direct gene transfer using particle gun and adequately exhibiting the possibility of stable transformation in Jatropha. This study has long-term implications in genetic engineering of J. curcas for desired traits.

2. Materials and methods 2.1. Plant material and culture conditions J. curcas seeds of CP-9 cultivar were used for regeneration and transformation. Mature decoated seeds of Jatropha were surface sterilized with 0.1% (w/v) mercuric chloride for 15 min and washed 4–6 times with sterile distilled water. Embryo explants were dissected out aseptically from endosperm and cotyledons removed carefully. Explants were pre-cultured for five days on optimized solid MS basal media (Murashige and Skoog, 1962) (pH 5.8) supplemented with 2.22 ␮M BA, 0.8% (w/v) agar and 3% (w/v) sucrose. All cultures were maintained under controlled laboratory conditions at 25 ± 2 ◦ C under a 16/8 h light/dark photoperiod with cool white 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 diameter of 25 mm on same media just before the bombardment. Plasmid pCAMBIA 1301 was isolated by using plasmid Miniprep kit (Qiagen, Germany) following manufacturer’s protocol. Transformation conditions were determined using the plasmid pCAMBIA 1301, which harbours the gus reporter gene and the selectable hptII gene, both controlled by the cauliflower mosaic virus (CaMV) 35S promoter. 2.2.1. Preparation of microcarriers Microcarriers (0.5 mg gold) coated with 1 ␮g of plasmid DNA and suspended in 50 ␮l absolute ethanol, were used as a standard for each bombardment. Gold microparticles were suspended in 1 ml 70% ethanol (v/v) by vigorous vortexing for 3–5 min followed by soaking for 15 min. Microparticles were washed 3 times with 1 ml sterile water by spinning for 30 s in a microfuge. After third wash, microparticles were suspended in sterile 50% glycerol and coated with plasmid DNA (pCAMBIA 1301) using CaCl2 (2.5 M) and spermidine (0.1 M) precipitation method. After 10 min incubation on ice, the supernatant was removed and pellet was washed with 70% (v/v) ethanol followed by washing with absolute ethanol. After washing, the particle DNA pellet was re-suspended in absolute ethanol for bombardments. Care was taken to ensure uniform particle distribution and minimize agglomeration. 2.2.2. Microprojectile bombardment Bombardments were done with biolistic gene gun (PDS 1000/He, Bio-Rad) under a vacuum of 27 in. of Hg, a 25 mm distance from rupture disc to macrocarrier and a 10 mm macrocarrier flight distance for all bombardments. The variables to be optimized included five rupture disc pressures (650, 900, 1100, 1350 and 1550 psi), four microprojectile travel distances (3, 6, 9, and 12 cm) and microcarrier size (gold particle size 0.6, 1.0 and 1.6 ␮m). Non-bombarded embryo axes and embryo axes bombarded with uncoated microcarriers were used as controls. 2.3. Selection and regeneration of transformants After bombardment, explants were kept in dark at 25 ◦ C for 24 h and then transferred to shoot induction medium (SIM), i.e. MS medium containing 3% sucrose, 0.8% agar and plant growth regulators 2.22 ␮M BA + 2.27 ␮M TDZ + 0.49 ␮M IBA. After 15 days the explants were transferred to selection medium (same as above) containing 5 mg L−1 hygromycin. For effective selection, the 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 GA3 ) with increasing concentration (6 and 7 mg L−1 ) of hygromycin. After three cycles of selection, putative transformed shoots were transferred for approximately 40–60 days to shoot elongation

0 72.7 100 57.6 ± 29.8 57.5 ± 9.14G 88.9 100 70.0 86.3E ± 8.8

About 110–200 mature embryo axes were bombarded per treatment; means in each column followed by same lower case and capital letters are significantly different for their respective data set according to Tukey’s HSD at P < 0.01 and P < 0.05 respectively.

43.9D ± 7.3 59.1 ± 5.7 66.1D ± 6.6 50.0 60.0 77.8 62.6 ± 8.1 33.3 75.0 100 69.4 ± 19.5

3 12 9

50.0 44.4 60.0 51.5E ± 4.6 66.7 66.7 100 77.8 ± 11.1

6 3

60.0 80.0 100 80 ± 11.6 73.9 ± 5.68y

1550 Microprojectile travel distance (cm)

1350 Size

10.0 44.5 66.7 40.4 ± 16.5

9 6

12

Mean ± SE Helium pressure (psi) Microcarrier

9

0 33.3 50.0 27.8A ± 14.7 54.6 66.7 80.0 67.1 ± 7.3

6 3

80.0 88.9 70.0 79.6A ± 5.5 53.2 ± 8.39F 0.6 ␮m Gold 1.0 ␮m Gold 1.6 ␮m Gold Mean ± SE Grand mean

0.6 ␮m Gold 1.0 ␮m Gold 1.6 ␮m Gold Mean ± SE Grand mean

100 100 100 100cB

22.2 30.0 33.3 28.5cd ± 3.3

9

72.7 80.0 88.9 80.5dB ± 4.7 66.7 70.0 72.7 69.8c ± 1.7 69.7 ± 7.98x 0 22.2 10.0 10.7b ± 6.4

6 3 12

10.0 10.0 22.2 14.0a ± 4.1 44.4 50.0 50.0 48.1ab ± 1.8 70.0 33.3 10.0 37.7 ± 17.5 27.7 ± 6.26xyFG

9 6 3 12

900 Microprojectile travel distance (cm)

650 Size

Helium pressure (psi) Microcarrier

Table 1 Cumulative effect of microcarrier size, helium pressure and target distance on transient gus expression.

0 54.6 60.0 38.2 ± 19.2

1100

12

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medium (SEM; MS + 3% sucrose, 0.8% agar + 2.22 ␮M BA) for further shoot elongation. For rooting, elongated shoots were transferred to root induction medium (RIM) containing half-strength MS basal salts, 1.47 ␮M IBA and 2% sucrose. After 4 weeks, plantlets with rooted shoots were transplanted into pots (5 × 10 cm) covered with transparent plastic lids and maintained under high humidity for 7–10 days, thereafter gradually exposed to culture room conditions followed by green house conditions. Established plantlets were transferred to pots for hardening. 2.4. Histochemical GUS assay Transient gus expression was assessed after 24 h of bombardment and randomly 20 transformed embryos per shot per plate were selected (Jefferson et al., 1987). Whole plantlets and leaves of transgenic lines (4–5-month-old after bombardment) were assayed for the constitutive expression of gus gene. GUS assay was done by incubating the tissues in freshly prepared GUS assay buffer (1 g L−1 X-Gluc with 0.05 M Na2 HPO4 , 0.5 mM K3 Fe(CN)6 , 0.5 mM K4 Fe(CN)6 , 10 mM EDTA and 0.1% (v/v) Triton X-100) for 12 h at 37 ◦ C. Thereafter tissues were destained with 70% alcohol to examine and count blue spots. Explants with at least one discrete blue region on the tissue were scored for GUS positive. 2.5. Molecular analysis 2.5.1. PCR amplification The genomic DNA of transformants was isolated using CTAB method (Doyle and Doyle, 1987). Transformation was confirmed by PCR with gus (reporter gene) specific primers (F: 5 -GAT CGC GAA AAC TGT GGA AT-3 and R: 5 -TGA GCG TCG CAG AAC ATT AC3 ) and hptII (hygromycin selection marker gene) specific primers (F: 5 -TTC TTT GCC CTC GGA CGA GTG-3 and R: 5 -ACA GCG TCT CCG ACC TGA TG-3 ) using initial denaturation temperature of 94 ◦ C for 10 min, subsequent 35 cycles of 94 ◦ C denaturation for 1 min, 60 ◦ C annealing for 1 min, 72 ◦ C extension for 1.5 min and final extension at 72 ◦ C for 7 min. 2.5.2. Southern hybridization Genomic DNA (20 ␮g) from transformants was digested with EcoRI, separated by electrophoresis in a 0.8% agarose gel and transferred onto a Hybond N+ membrane (Amersham Pharmacia, UK) by capillary method using alkaline transfer buffer (0.4N NaOH with 1 M NaCl). The membrane was air-dried and DNA was fixed to the membrane by UV cross-linking using 56 mJ cm−2 energy for 1 min in a UVC 500 cross-linker (Amersham Biosciences, UK). Blot was hybridized with PCR-generated probe for hptII gene labeled with DIG-11-dUTP, amplified from plasmid pCAMBIA 1301 using 0.1 mM DIG-11-dUTP, 1.9 mM dTTP and Taq DNA polymerase, following manufacturer’s user guide (Roche, Germany). Purified pCAMBIA 1301 and PCR amplified hptII gene served as a positive control while DNA from non-transformed plant as a negative control. Prehybridization and hybridization were carried out at 68 ◦ C overnight in DIG EasyHyb buffer solution (Roche, Germany). The membrane was then washed 2–3 times at room temperature for 5 min in 2× SSC, 0.1% SDS, and twice for 15 min in 0.2× SSC, 0.1% SDS at 68 ◦ C. The hybridized membrane was detected by using CDPStar chemiluminescent as substrate, following manufacturer’s user guide (Roche, Germany) and signals were visualized on X-ray film after 30 min. 2.6. Statistical analysis Each treatment consisted of at least two plates and was replicated thrice. Frequency of GUS activity was calculated as number

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a

0.6 μm Gold 1.0 μm Gold 1.6 μm Gold Mean Poly. (Mean)

100

Transie ent gus expression (%)

90 80

f LSZ

e JQS mt

70 60

y = -1.402x2 + 13.89x + 30.12 R² = 0.248

gn u

ip w A

bcd

B

hov

kry

a

50 40 ef

30

bgh ik acmn JL o pr Q S

dtuvwy XZ

AB

20 10 0 650 psi

900 psi

1100 psi

1350 psi

1550 psi

b

40

Frequency of Sho oots survival (%)

Helium Pressure

35

y = -0.959x2 + 1.872x + 15.36 R² = 0.934

30

0.6 μm 1.0 μm 1.6 μm Mean Poly. (Mean)

25 20 15 10 5 0 650 psi

900 psi

1100 psi

1350 psi

1550 psi

Helium Pressure Fig. 1. Effect of microcarrier size and helium pressure on (a) transient gus expression and (b) survival of shoots after 3rd round of selection. Means ± SE followed by same capital case and lower letters are significantly different for their respective data set according to Tukey’s HSD at P < 0.01 and P < 0.05 respectively. Trend line indicates polynomial regression (order value 2) for grand mean value.

of embryos showing gus expression to the total number of explants stained after bombardment and is expressed as percentage. Data on the number of embryo axes showing transient gus expression was subjected to analysis of variance (ANOVA) for analysis to determine differences (Sokal and Rohlf, 1995) and were expressed as mean ± SE. A Tukey’s HSD multiple comparison of mean test was used when significant differences were found and P < 0.01 was considered as significant. 3. Results Decotyledonated embryos were elongated up to 0.5 cm with distinct proliferated meristematic regions within 5 days of incubation on medium (MS basal of pH 5.8 + 0.8% (w/v) agar + 3% (w/v) sucrose + 2.22 ␮M BA). The elongated embryos were excised and microprojectile bombardment mediated transformation was optimized on these embryonic meristematic tissues. For assessment of the effect of microcarrier size, helium pressure and microprojectile travel distance during bombardment, frequency of transient gus expression and survival of putative transformants after third cycle of selection were taken into consideration. 3.1. Microcarrier size vs. helium pressure Frequency of transient gus expression was 43.9 ± 7.3, 59.1 ± 5.7 and 66.1 ± 6.6, however, frequency of shoot survival after three rounds of hygromycin selection was 13.2 ± 2.16, 16.14 ± 2.95 and 1.95 ± 0.7 for 0.6, 1.0 and 1.6 ␮m gold microcarriers (Table 1 and S1). Transient gus expression was significantly increased

(P < 0.05) while frequency of shoot survival decreased (P < 0.01) with increase in microcarrier size. Overall, optimum microcarrier size was observed 1.0 ␮m while comparing transient gus expression and frequency of shoot survival (S1). Like microcarrier size, transient gus expression was increased while frequency of shoot survival decreased concomitantly with helium pressure (Table 1 and S2). Maximum transient gus expression was observed at 1100 and 1350 psi for 0.6 and 1.0 ␮m microcarrier. Apart to this, significant transient gus expression was also observed at 1550 psi for 1.6 ␮m microcarrier (Fig. 1a). Optimum transient gus expression was observed at 1100 and 1350 psi He pressure (Table 1 and Fig. 1a). Maximum survival of shoots was observed at 650 and 900 psi He pressure for 0.6 and 1.6 ␮m while 1100 psi for 1.0 ␮m microcarriers (Table 2 and Fig. 1b). Overall optimum He pressure was observed 1100 and 1350 psi and microcarrier size 1.0 ␮m independent to microprojectile travel distance while comparing transient gus expression and frequency of shoot survival (Fig. 1a and 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 of 3, 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 expression and frequency of shoot survival (Fig. 2a and b), optimum travel distance was observed 9 and 12 cm and microcarrier size 1.0 ␮m independent to He pressure.

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

3.3. Helium pressure vs. microprojectile travel distance The mean transient gus expression was 53.18 ± 12.11, 27.63 ± 9.1, 69.7 ± 15.09, 73.9 ± 7.68 and 57.5 ± 6.19 for 650, 900, 1100, 1350 and 1550 psi He pressure respectively (Table 1

71

and Fig. 3a). Frequency of shoot survival declined with increase in target distance (Table 2 and Fig. 3b) while comparing transient gus expression and frequency of shoot survival, optimum travel distance was observed 9 and 12 cm with He pressure 1100 and 1350 psi respectively independent to microcarrier size.

Table 2 Effect of microcarrier size, helium pressure and target distance on transformation efficiency. Gold particle size

0.6 ␮m

Helium pressure (psi)

650

900

1100

1350

1550

1.0 ␮m

650

900

1100

1350

1550

1.6 ␮m

650

900

1100

1350

1550

Microprojectile travel distance (cm)

No. of explants bombarded

Relative frequency of transient gus expression (percentile)

Selection I

Selection II

Selection III

3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12

120 127 133 123 155 143 172 149 152 141 125 156 131 129 140 142 121 126 136 139

35.69 33.43 31.48 10.0 36.9 34.49 32.68 30.87 38.10 34.04 36.6 32.08 40.60 38.71 36.0 32.98 52.71 51.66 33.58 50.0

92.5 98.4 97.7 91.1 82.6 95.1 87.8 91.9 75.0 79.4 96.8 98.1 71.8 95.3 87.1 93.0 70.2 75.4 72.1 73.4

42.5 50.4 55.6 50.4 41.3 40.6 53.5 53.7 28.9 34.8 37.6 41.0 18.3 14.7 29.3 26.8 4.1 5.6 19.1 19.4

24.2 24.4 20.3 21.1 21.3 20.3 22.7 26.8 19.1 16.3 13.6 15.4 2.3 1.6 3.6 1.4 0 0 3.7 5.8

3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12

192 188 187 195 168 177 175 164 173 156 188 181 178 193 192 174 164 175 192 190

50.90 52.11 48.04 14.76 38.25 35.30 35.09 32.08 42.92 40.06 68.07 35.54 47.29 41.42 36.30 64.00 58.28 54.97 36.75 35.39

89.1 91.5 92.5 95.9 83.3 88.7 95.4 97.6 89.6 91.0 98.4 99.4 86.5 91.2 89.1 97.7 71.3 76.0 72.4 77.4

40.1 44.1 48.7 45.1 48.2 52.0 46.3 53.7 35.3 32.1 58.0 47.5 19.7 17.1 31.8 49.4 2.4 8.0 1.6 2.1

23.4 25.0 19.8 27.2 12.5 11.3 13.7 12.8 10.4 21.2 44.7 32.0 0 8.3 19.8 39.1 0 0 0 1.6

3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12 3 6 9 12

178 197 163 184 187 193 187 175 173 185 181 178 199 192 158 163 144 147 132 108

71.08 68.68 64.61 59.64 73.34 56.18 50.75 44.88 76.96 52.11 57.38 45.78 64.06 61.90 55.27 48.95 100 79.82 62.05 53.62.

80.9 78.7 92.6 93.5 86.1 87.0 88.8 90.9 71.1 81.6 91.7 88.8 70.9 72.9 78.5 82.8 67.4 68.7 74.2 84.3

32.0 39.1 33.7 32.1 11.2 14.5 13.4 10.3 1.7 1.6 8.3 6.7 2.0 3.1 4.4 5.5 0 0 0 0

0 1.5 4.3 11.4 0 5.7 6.4 5.7 0 0 1.7 2.2 0 0 0 0 0 0 0 0

Frequency of shoot survival (%)

Subculture was done at 3weeks interval onto respective medium containing 5, 6 and 7 mg hygromycin/l for selections I, II and III, respectively. Optimized conditions are shown in bold numerical.

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a

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

100

Transient gus expression (%)

90

0.6 μm Gold 1.0 μm Gold 1.6 μm Gold

bde

Mean

80

Poly. P l (Mean) (M )

70

a

c

60

b

50

e

40 30

acd

20 10 0 3 cm

6 cm

9 cm

12 cm

b

35

hoots survival (%) Frequency of Sh

Microprojecle travel distance (cm)

30

0.6 μm Gold 1.0 μ μm Gold 1.6 μm Gold Mean Poly. (Mean)

y = 0.097x2 + 1.555x + 5.807 R² = 0 992 0.992

25

eij cg

20 15

a

dh

bf

10 5

abc de

fghi

j

0 3 cm

6 cm

9 cm

12 cm

Microprojecle travel distance (cm) 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 letters are 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 letters are significantly different according to Tukey’s HSD at P < 0.01. Trend line indicates polynomial regression (order value 2) for grand mean value.

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

3.4. Tissue culture and histochemical GUS assay Transformed embryos per shot per plate were selected randomly after 24 h of bombardment and transient gus expression

73

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

Fig. 4. Microprojectile bombardment-mediated transformation, selection, regeneration and GUS assay. Transient gus expression on (a) embryo axis, (b) whole embryo, (c) radicle part of embryo and (d) inner side (transverse section) of embryo axis after 24 h of bombardment. Five days elongated embryo ready to transform was shown as insight (a). Selection of transformants (e) 1st round (28 days old), (f) 2nd round (48 days old) and (g) 3rd round (68 days old) on hygromycin. Regeneration of transformants; (h) shoot regeneration (90 days old), (i) shoot elongation (140 days old), (j) rooting (170 days old) and (k) hardening of transgenic plant (3-month-old after hardening; plant shown in insight is 200 days old). GUS assay of (l) non-transformed leaf, (m) whole plantlet and (n) leaf of transgenic plant.

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The transformants were transferred to selection medium (SIM) containing 5 mg L−1 hygromycin for 1st round of selection for 21 days (Fig. 4e), thereafter transferred to shoot regeneration medium (SRM; MS + 3% sucrose + 0.8% agar + 2.22 ␮M BA + 0.49 ␮M IBA + 1.45 ␮M GA3 ) containing 6 mg L−1 hygromycin for 2nd round of selection for 21 days (Fig. 4f). For effective selection, regenerated transformants were transferred to same medium containing 7 mg L−1 hygromycin for 21 days for 3rd round of selection (Fig. 4g). During selection on medium with hygromycin, non-transgenic tissues gradually turned brown, while putative transformed sectors remained green and exhibited slow growth (Fig. 4e–g). The maximum transformation efficiency 44.7% was observed with 1 ␮M gold, 1100 psi He pressure and 9 cm travel distance followed by 39.1% with 1350 psi He pressure and 12 cm travel distance (Table 2). Transformation efficiency is calculated as number of PCR positive plants survived after third round of selection with respect to total embryos bombarded. After three cycles of selection, putative transformed shoots were transferred for approximately 40–60 days to the MS medium containing 3% sucrose, 0.8% agar and 2.22 ␮M BA for further shoot elongation (Fig. 4h and i) and subsequently transferred to root induction medium (1/2MS + 2% sucrose + 0.8% agar + 1.47 ␮M IBA) for approximately 28 days (Fig. 4j). Putative transgenics with rooted shoots were transplanted into pots for hardening (Fig. 4k). Meanwhile whole plantlets and leaves were assayed for the constitutive expression of gus gene using nontransformed leaf as control (Fig. 4l–n). 3.5. Molecular analysis of transgenic plants The confirmation of genetic transformation was based on the presence of reporter gene gus and selectable marker gene hptII by PCR amplification of expected bands of sizes 1.28 and 0.96 kb, respectively (Fig. 5a and b). Randomly 3-month-old putative transgenic plants transformed with optimized conditions and survived after three rounds of selection were selected for the molecular analysis. All plants tested were observed positive for both gus and hptII genes. Southern analysis was done following PCR confirmation of gus and hptII genes for the same transformants. Southern analysis revealed detectable signals in PCR positive plants for hptII gene (Fig. 5c). Generally, transformants showed single copy while two transgenic plants contained multiple (2 and 3) copies of the introduced gene. 4. Discussion Gene introgression by particle bombardment is most efficient and consistent, genotype independent versatile physical process with no biological constraint (Altpeter et al., 2005). A simple and efficient microprojectile mediated genetic transformation method in Jatropha was established with 44.7% transformation efficiency (Fig. 6). Different physical parameters need to be carefully examined in particle bombardment that could enhance transient GUS expression and lead to stable integration of the introduced genes (Sailaja et al., 2008). In this study, different physical parameters such as microcarrier size, velocity of particle delivery and microprojectile travel distance were optimized individually and incombination considering the frequency of transient gus expression and survival of putative transformants. Microprojectile bombardment with embryonic axis as target tissues has been used for the production of successful transgenic lines in many plants, i.e. soybean (McCabe et al., 1988; Rech et al., 2008), cowpea (Ivo et al., 2008), peanut (Brar et al., 1994; Livingstone and Birch, 1999), castor (Sailaja et al., 2008) and corn (Lowe et al., 2009). Microprojectile bombardment is independent to any kind of target tissue but in this study embryo axes, pre-cultured for 5 days on MS medium

Fig. 5. Molecular analysis of transgenic plants. (a) PCR amplification of the hptII gene (963 bp), (b) gus gene (1286 bp) and (c) southern analysis of randomly selected putative transgenic plants transformed with optimized conditions. Lane ML: molecular weight marker ladder; lane ␭M: ␭DNA EcoR1/HindIII double digest molecular weight marker; lane PC1: PCR amplified hptII gene (positive control); lane PC2: PCR amplified gus gene (positive control); lane PC3: pCAMBIA 1301 (positive control); lane NTC: non-transformed plantlet (negative control), and lanes J1–J8: putative transgenic plants.

were taken as both castor and Jatropha belong to Euphorbiaceae family and embryo axis was found suitable for stable genetic transformation in castor (Sailaja et al., 2008) and regeneration (Sujatha and Mukta, 1996). Embryo was pre-cultured for 5 days to maximize the probability of stable transformation as actively dividing cells have ability to survive and grow under stress imposed during bombardment process (Sailaja et al., 2008). Frequency of transient gus expression was significantly increased (P < 0.05) and frequency of shoot survival decreased (P < 0.01) with an increase in particle size. Similarly, transient gus expression was increased while frequency of shoot survival decreased concomitantly with helium pressure. High pressure and large microcarriers penetrated into deep cell layers and integrated with genome, hence more transient gus expression was observed.

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Decoated mature seeds Sterilization with 0.1% HgCl2 for 15 min Embryo axes excised and cultured on MS medium with 2.22 μM BA d di l removedd 5 days, radicles 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 Hygromycin 21 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 selection Subculture of regenerated shoots to shoot elongation medium (SEM) containing 2.22 μM BA 28 days Subculture to SEM for further elongation of shoots 28 days Transfer to root induction medium containing ½MS and 1.47 μM IBA 28-35 days Rooted plants transplanted and acclimatized in plastic pots 28-35 days T f tto pots t iin fi ld conditions diti Transfer field

Fig. 6. Schematic representation of the complete protocol for microprojectile bombardment mediated genetic transformation and regeneration of J. curcas using embryo axes.

Simultaneously, it also imposed injury leading to decrease in probability of shoot survival. The optimum He pressure was observed 1100 and 1350 psi and microcarrier size 1.0 ␮m independent to microprojectile travel distance, while comparing transient gus expression and frequency of shoot survival. In this combination, gene introgression was efficient for transient gus expression leading to maximum shoot survival. Decline in transient gus expression with increase of travel distance in combination with microcarrier size independent to He pressure could be due to deceleration of the microprojectile velocity. However, opposite result was observed in combination with He pressure independent to microcarrier size because of acceleration of microcarrier with high pressure. While comparing transient gus expression and frequency of shoot survival, optimum travel distance was observed 9 and 12 cm for He pressure 1100 and 1350 psi respectively, independent to microcarrier size. The genetic transformation efficiency was determined at different microcarrier size, He pressure and target distance both in terms of transient gus expression and shoot survival. The study revealed that transient gus expression is not the key to analyze transformation efficiency. In spite of a very high frequency of transient gus expression at 1550 psi for 1.6 ␮m microcarrier, the frequency of surviving explants drastically declined during selections because high helium pressure increased particle acceleration and subsequent target tissue penetration leads to injury by DNA coated microcarriers.

Overall optimum parameters were observed for microcarrier size 1.0 ␮m, He pressure 1100 and 1350 psi with target distance 9 and 12 cm respectively by comparing transient gus expression and frequency of shoot survival. The efficiency of transformation obtained through particle gun gene transfer in the present study is 44.7% and higher than those reported earlier for Jatropha (Li et al., 2008; Kumar et al., 2010; Purkayastha et al., 2010). The transformation efficiencies through Agrobacterium-mediated transformation were 13% with cotyledon disc (Li et al., 2008) and 29% with leaf explants (Kumar et al., 2010), however, Purkayastha et al. (2010) transformed Jatropha using shoot apices by particle bombardment but transformation efficiency was not reported. Higher efficiency of transformation through particle gun bombardment could probably be due to the higher number of explants tried with varying individual and in-combination physical parameters. High transformation efficiency over previous methods makes optimized protocol (Fig. 6) efficient and thus has the potential to facilitate the genetic modification for trait improvement. The optimized parameters were efficiently used previously for evergreen coniferous tree Norway spruce (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 browning is a typical feature of callus cultures derived from the hypocotyl of J. curcas (He et al., 2009) and during selection on hygromycin

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medium, non-transgenic tissues gradually turned brown, while putative transformed sectors remained green and showed slow growth. Brown tissues result in decreased regenerative ability, poor growth and subsequent death. Histochemical GUS assay after the final selection of transformed lines, showing constitutive expression of gus gene, confirmed the efficient integration of gene which is also evident with the PCR amplification of both gus and selectable marker hptII gene. Generally, in microprojectile bombardment mediated genetic transformation, multiple copies of inserted genes are reported (Altpeter et al., 2005), however, in this work, mostly (75%) single copy of insertion was observed by southern analysis, confirming the efficacy of the present method. 5. Conclusion ANOVA reveals highly significant effects of microcarrier size, helium pressure, target distance and their interaction on transient gus expression and frequency of transformants survival. Despite the increase in frequency of transient gus expression with increase in helium pressure and microcarrier size, there was a reduction in the frequency of surviving shoots and shoots failed to survive after third cycle of selection in some combinations of parameters. The effect of helium pressure and its interaction with target distance on the frequency of shoot survival was highly significant. Significant and polygonal correlations of helium pressure and target distance on the frequency of gus expression and shoot survival of putative transformants were found. An efficient microprojectile bombardment mediated genetic transformation and plant regeneration protocol was established for J. curcas using embryo axes (Fig. 6). The results of the present investigation adequately exhibit the possibility of stable transformation of Jatropha through direct gene transfer method with 44.7% transformation efficiency. The optimized protocol will be very useful for introduction of desired gene in biofuel plants for the trait improvement in future. Acknowledgement The financial support received from CSIR, New Delhi (Network Project NWP-20) is thankfully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.indcrop.2010.09.002. References Altpeter, F., Baisakh, N., Beachy, R., Bock, R., Capell, R., Christou, P., Daniell, H., Datta, K., Datta, S., Dix, P.J., Fauquet, C., Huang, N., Kohli, A., Mooibroek, H., Nicholson, L., Nguyen, T.T., Nugent, G., Raemakers, K., Romano, A., Somers, D.A., Stoger, E., Taylor, N., Visser, R., 2005. Particle bombardment and the genetic enhancement of crops: myths and realities. Mol. Breed. 15, 305–327. Batista, D., Fonseca, S., Serrazina, S., Figueiredo, A., Pais, M.S., 2008. Efficient and stable transformation of hop (Humulus lupulus L.) var., Eroica by particle bombardment. Plant Cell Rep. 27, 1185–1196. Berchmans, H.J., Hirata, S., 2008. Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids. Bioresour. Technol. 99, 1716–1721. Bohorova, N., Zhang, W., Julstrum, P., McLean, S., Luna, B., Brito, R.M., Diaz, L., Ramos, M.E., Estanol, P., Pacheco, M., Salgado, M., Hoisington, D., 1999. Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos. Theor. Appl. Genet. 99, 437–444. Brar, G.S., Cohen, B.A., Vick, C.L., Johnson, G.W., 1994. Recovery of transgenic peanut (Arachis hypogea L.) plants from elite cultivars utilizing ACCELL technology. Plant J. 5, 745–753. Chhetri, A.B., Tango, M.S., Budge, S.M., Watts, K.C., Islam, M.R., 2008. Non-edible plant oils as new sources for biodiesel production. Int. J. Mol. Sci. 9, 169–180. Cho, M.J., Yano, H., Okamoto, D., Kim, H.K., Jung, H.R., Newcomb, K., Le, V.K., Yoo, H.S., Langham, R., Buchanan, B.B., Lemaux, P.G., 2004. Stable transformation of

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