Sonication and anti-necrotic agents improved agrobacterium-mediated

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An improved method of transgenic plant production of seedless grapevine with anti-fungal genes, chitinase and glucanase was developed. Embryogenic cultures of grapevine cv. Crimson Seedless initiated from in vitro leaves and matured somatic embryos were used as explants for Agrobacterium-mediated transformation studies. Sonication of somatic embryos and incorporation of thiol compounds and other anti-necrotic agents in the co-cultivation medium significantly improved the transformation efficiency of somatic embryos. Sonication of embryos suspended in Agrobacterium broth at a cell density of 0.5 OD600 for 3 s was found optimum irrespective of the binary vector used for transformation. Transformation efficiency (%) increased by 4 fold (chitinase) ­to 7.5 fold (glucanase) on incorporation of phenylalanine (2 mg/l) and sodium thiosulphate (20 mg/l), respectively, in the solid co-cultivation medium as compared to the control. The integration of chitinase and glucanase genes was confirmed by gene specific PCR, gene sequencing and southern blot analysis. The transgenic plants showed enhanced activity of chitinase and glucanase compared to untransformed control. Transgenic plants of Crimson Seedless with integrated anti-fungal genes were transferred to soil-sand-cocopeat (1:1:1) mixture and hardened plantlets were established in the greenhouse. The transgenic plants showed varied degree of enhanced tolerance to Downy mildew fungus under laboratory conditions.

Keywords: Agrobacterium; anti-oxidants; fungal tolerance; grapevine; sonication

Introduction

Diseases in grapevine cause growers invest a lot of money and labor on various techniques to reduce crop losses. Vines with improved disease resistance would be advantageous, especially if other promising traits of the cultivar were not altered. Reduction of fungicide sprays by even one or two per year would cut the cost of production to a great amount and also benefit the environment. Moreover improvement of grapevine for disease resistance through classical breeding is cumbersome and time taking process. Alternatively, genetic engineering approaches may be used to introgress disease resistance through introduction of genes encoding high-value recombinant proteins involved in disease tolerance mechanism. Isolation of genes encoding a class of proteins (chitinases, glucanases, RIPs etc.) with anti-fungal activity, called pathogenesis related (PR) proteins has opened up new era of developing plant resistance to fungal diseases. The proteins, chitinase and glucanase possess antifungal activity by cleaving the main components of fungal cell walls, chitin and β-glucan, respectively. Genes encoding these proteins were over expressed in model plant tobacco which showed enhanced tolerance to fungal pathogens (Zhu et al. 1994). Attempts were also made to introduce these anti-fungal genes in to the grapevine cvs. Dornfelder, Riesling and Muller-Thurgau (Bornhoff et al. 2005).

Efficient production of transgenic plants through Agrobacterium-mediated transformation is reported to be limited by the tissue necrosis and cell death due to oxidative burst caused by Reactive Oxygen Species (ROS) (Gustavo et al. 1998). These ROS can also lead to the production of PR proteins (Mehdy 1994), which may inhibit the potential of Agrobacterium to colonize and transfer its T-DNA in to the target cells. The activity of oxidative burst can be suppressed by the addition of anti-oxidants such as ascorbic acid, citric acid, cysteine, polyvinylpyrrolidone (PVP), dithiothreitol (DTT) and anti-bacterial agents like silver nitrate. Some of these compounds are known to scavenge ROS, thereby quenching the oxidative burst thus improving the efficiency of Agrobacterium-mediated transformation (Perl et l. 1996; Olhoft et al. 2001; Kuta and Tripati 2005; Li et al. 2009). Though grapevine has been transformed and transgenic plants have been developed so far, the success is mostly restricted to few species and cultivars. Till date, there is no report on biotechnological improvement of grapevine cv. Crimson Seedless for fungal disease resistance. In this paper we report efficient production of transgenic plants of Crimson Seedless with anti-fungal genes, chitinase and glucanase using sonication and anti-necrotic agents.

Materials and Methods

Initiation of embryogenic cultures

Embryogenic cultures of grapevine cv. Crimson Seedless were initiated from in vitro leaves of the cultivar collected from multiple shoots proliferated on MS medium (Murashige and Skoog 1962) supplemented with N6-benzyladenine (BA) (8.89 µM). For callus induction, in vitro leaves were cultured on Callus Induction Medium (CIM) composed of half strength MS (½MS, containing half the concentration of major and minor nutrients and MS vitamins) supplemented with BA (4.44 µM), naphthoxyaceticacid (NOA, 4.95 µM) and phenylalanine (5.0 mM) with regular sub cultures at 4 weekly interval. After 5th sub culture, embryogenic callus was shifted to ½MS medium devoid of growth regulators for induction of somatic embryos. Somatic embryos induced repetitive embryogenesis on shifting to fresh medium. Mature torpedo to cotyledonary stage somatic embryos were used as the target material for the Agrobacterium-mediated transformation. Sucrose at 30 g l-1 was added as carbon source to all the media gelled with agar (0.65%, w/v) and the media were autoclaved at 121oC for 20 min after adjusting the pH to 5.8. All the cultures were incubated at 25±2oC and under continuous dark for induction and proliferation of somatic embryos, and under 16 h photoperiod for germination and conversion of somatic embryos.

Agrobacterium strain and plasmids

Agrobacterium tumefaciens strain LBA4404 carrying modified binary vectors pCAMBAR.chi.11 and pCAMBAR.638 harboring anti-fungal genes, 'chitinase' and 'glucanase', respectively, both under the control of maize ubiquitin promoter was used for the transformation experiments (Fig. 1A,B). The two recombinant plasmids also contained bar and hpt genes as plant selectable markers under the control of CaMV35S promoter.

Transformation and generation of transgenic plants

Single colony of the Agrobacterium was picked up and cultured overnight in 5 ml YEB (sucrose 5 g l-1, beef extract 5 g l-1, bacto-peptone 5 g l-1, yeast extract powder 1 g l-1 and magnesium sulphate 490 mg l-1, pH 7.2) containing kanamycin 50 mg l-1 and streptomycin 50 mg l-1 on shaking incubator at 200 rpm at 28oC. One ml of the overnight culture was transferred to 50 ml YEB with antibiotics and cultured at the same culture conditions. After 8-12 h of culture, the OD of the culture was measured using spectophotometer with absorption at 600 nm. The bacterial broth was taken in to a centrifuge tube and the cells were pelleted at 4000 rpm for 10 min at 4oC. The supernatant was discarded and the pellet was washed and re-dissolved in ½MS liquid medium to a required OD600 (0.2 - 0.75).

Mature torpedo to cotyledonary stage somatic embryos of Crimson Seedless capable of repetitive somatic embryogenesis were used for the transformation studies (Fig. 3A). Somatic embryos were treated with Agrobacterium cell suspension for 30 min. Then these embryos were mildly blotted on sterile filter paper and co-cultured on antibiotic free ½MS medium for 24 - 72 h in dark at 25oC. After co-cultivation, the embryos were washed 2-3 times with sterile ½MS liquid medium followed by one wash with ½MS + cefotaxime (250 mg l-1) and the embryos were blotted on sterile filter paper. There after, somatic embryos were cultured on ½MS + BA (1 M) + cefotaxime (250 mg l-1) for two weeks. Later they were shifted to Selection Medium-1 [SM1, ½MS + BA (1 M) + hygromycin (5 mg/l)] and cultured for two weeks. Then the embryos were shifted to Selection Medium-2 [SM2, ½MS + BA (1 M) + hygromycin (10 mg l-1)] for final selection. Putatively transformed embryos on SM2 induced repetitive somatic embryogenesis on same medium and matured somatic embryos were germinated on Woody Plant Medium (WPM) (Llyod and McCown 1981) supplemented with BA (4.44 µM), indole-3-butyric acid (IBA, 0.49 µM) and hygromycin (10 mg l-1). Germinated and converted embryos were transferred to individual test tubes and after 1 week they were planted in pots (10 cm diameter and 15 cm deep) containing soil-sand-peat (1:1:1) mixture and hardened according to the procedure described earlier (Nookaraju et al. 2008).

Influence of sonication

To investigate the influence of sonication on transformation efficiency, somatic embryos were suspended in a 1.5 ml microfuge tubes containing Agrobacterium suspension at an OD600 of 0.2 - 0.75 and were sonicated at 60 KHz for a period of 1 - 10 s in a bath sonicator (Bransonic Ultrasonic Corporation, USA). Immediately after sonication, the tubes were placed in ice for a while and the embryos were co-cultivated for 48 h as mentioned earlier. Somatic embryos after sonication were observed for their surface properties under Environmental Scanning Electron Microscope (ESM, JOEL 11008 attached with EDAX) at 4oC temperature, 4.19 Torr chamber pressure at an accelerating voltage of 30 KV and photographed.

Influence of anti-necrotic agents

To investigate the influence of various anti-oxidants / anti-necrotic agents on Agrobacterium-mediated gene transfer, anti-necrotic agents such as myo-inositol (100 mg l-1), silver nitrate (0.5-5.0 mg l-1), citric acid (5-50 mg l-1) or phenylalanine (1-3 mg l-1) and thiol compound like L-cysteine (100-800 mg l-1) or sodium thiosulphate (5-20 mg l-1), were supplemented individually in ½MS medium used for co-cultivation of somatic embryos. Before co-cultivation, somatic embryos were treated with Agrobacterium suspension at an OD600 of 0.5 for 30 min. After 72 h of co-cultivation, the embryos were washed and transferred to selection medium as mentioned earlier.

Analysis of transformants

Survival on selection medium

Independent primary transformants (secondary somatic embryos formed on SM2) were counted using stereomicroscope (Leica, model MZ125, Japan). Transformation efficiency was calculated at 12 weeks after culture on SM2 based on the number of secondary somatic embryos formed on SM2 out of total number of embryos used for transformation. Each experiment was repeated for a minimum of three times, the data were analyzed using analysis of variance (ANOVA) and treatment means were compared by Duncan's multiple range test (Duncan 1955).

DNA extraction and PCR

Genomic DNA from putative transformants was isolated by homogenizing 50-100 mg of cotyledonary leaves of germinated embryos using CTAB method (Lodhi et al. 1994). Plasmid DNA from the Agrobacterium was isolated using standard alkaline lysis method (Sambrook et al. 1989). Putative transformants were screened for the presence of chitinase (1.4 kb) and glucanase (1.6 kb) genes using the sequence specific primers. The primers used were: Ubi F, 5′- CCCTGCCTTCATACGCTAT-3′ (forward primer in the intron region of ubiquitin promoter) and PolyA R, 5′- GGAATTCAAGCTTCATC GAGCTCGGTA-3' (reverse primer in the CaMVpolyA). PCR was performed in a 25 μl reaction mixture containing 50 ng of DNA as template, 1X Taq DNA polymerase buffer, 400 μM each dNTPs, 10 pmol of each primer and 0.5U of Taq DNA polymerase. DNA amplifications were performed in a thermal cycler (Mastercycler personal, Eppendorf, Germany) using the programme: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 62.4°C for 1 min and extension at 72°C for 1.5 min with a final extension for 5 min at 72°C. The amplification products were visualized on 1% w/v agarose gel stained with ethidium bromide (0.5 μg ml-1).

Southern blotting

Southern blot analysis was performed according to the standard procedure (Sambrook et al. 1989). Approximately 20 mg of genomic DNA isolated from transgenic and untransformed control plants was double digested with XhoI enzyme to confirm the integration chitinase and glucanase genes, and single enzyme digestion with BstXI to detect the copy number as well as integration of the genes. The digested DNA was electrophoresed on 1% agarose gel and transferred onto Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Piscataway, N.J.) by capillary blotting method. XhoI fragments containing hygromycin phosphotransferase (hpt) gene from the either of the plasmids was radio-labeled with α32p by standard random prime labeling kit (Amersham Pharmacia Biotech, Piscataway, N.J.). After overnight hybridization, the membranes were washed with 2xSSC containing 0.1% SDS for 5 min followed by washing with 0.2xSSC containing 0.1% SDS for 10 min and blots were developed on X-ray films using the standard method.

Semi-quantitative and quantitative RT-PCR

Total RNA was extracted from the leaves of transgenic as well as untransformed control plants using TRI reagent (Sigma, USA). First-strand cDNA was synthesized using SuperScript Reverse Transcriptase (Invitrogen, USA). In order to distinguish the transgene-specific transcripts, a forward primer in the first exon of the ubiquitin promoter, 5′-CGTGTTGTTCGCAGCGCACAC- 3′, and a reverse primer in the CaMV polyA fragment, 5′-GCTCAACACATGAGCGAAACCC-3′, were used in the RT-PCR reaction. The RT-reaction was performed in a thermal cycler with PCR conditions as above with annealing at 64.7°C for 1 min. The transgene products were resolved on a 1.6% w/v agarose gel. Real time PCR was carried out using ABI Prism 7700 sequence detector (Applied Biosystems). The amplification of chitinase and β-1,3-glucanase was carried out using cDNA specific primers (Chi: 5'-TGC GGC TCC ACC TCC GAT TAC T-3' and 5'-GCG TCG TAG GTG TAG AAG CCC TTA-3'; Glu: 5'-TTG GAG TGC TTC TGG GAT CCA TTC-3' and 5'-GTC GAT GAT GAG GTC GAT GTT GCT-3'). The PCR was performed using SYBR green PCR kit (Bio-Rad, USA). Actin was used as an internal control. Comparative threshold (Ct) values were normalized to actin control and compared to obtain relative expression levels.

Enzyme assay

For enzyme studies, 50 mg of tissue from transformed and control embryolings was homogenized in 20 mM citric acid buffer (pH 6.8) in pre-chilled eppendorf tubes. The homogenate was centrifuged at 15000 rpm for 15 min at 4oC and the supernatant was used as crude enzyme extract. For chitinase assay, 2 mL of 50 mM citric acid (pH 6.8) containing 20 mg of carboxymethyl chitin (Himedia India Ltd, Mumbai, India) was mixed with 1 mL of the crude enzyme solution, incubated with shaking at 37°C for 1 h and the reaction was stopped by the addition of 1 mL of trichloroacetic acid. After centrifugation at 15000 rpm for 10 min, the concentration of reducing sugars in the supernatant was measured by the Schales method. One unit of enzyme is defined as the amount of chitinase that produces 1 µmol of reducing sugars and expressed as N-acetyl- D-glycosamine min-1.

Similarly, β-1,3-glucanase activity estimated by the method of Akiyama et al. (1998). The reaction mixture, containing 0.25% barley 1,3-β-glucan (Sigma G6513), 50 mM sodium acetate pH 5.0, and 10 µg crude protein from leaves of control and transgenic plants in a total volume of 100 µl, was incubated at 37oC. The reaction was stopped after 10 min by adding 300 µl of 1% p-hydroxybenzoic acid hydrazidein 0.5 M NaOH (Lever 1972) and boiling for 5 min, and the increase in reducing sugar determined by measuring absorbance at 405 nm. One unit of glucanase was defined as the amount of enzyme that liberated reducing sugar corresponding to 1 µmol glucose per minute under the stated conditions.

In vitro leaf disc assay for fungal tolerance

In vitro leaf disc assay was performed according the protocol described earlier (Brown et al. 1999). Leaf discs were excised from uninfected leaves using a cork borer of 2.5 cm in diameter. Leaf disks are laid on abaxial surface on filter paper saturated with distilled water in Petri dishes. The spore suspension of downy mildew extracted from oil spots was used for infecting the leaf discs. An evaluation was made at seventh day after infection. The disease severity was measured in terms of percentage inhibition of growth of fungal hyphae.

Results

In preliminary studies to standardize Agrobacterium cell density and co-cultivation period for efficient transformation of mature somatic embryos of Crimson Seedless, it was observed that transformation efficiency (as assessed by PCR) was maximum when the explants were treated with Agrobacterium at a cell density of 0.5 OD600 and co-cultivated for a period of 48 h (data not shown). Agrobacterium cell densities higher than 0.5 OD600 or co-cultivation longer than 48 h resulted in severe growth of Agrobacterium on the explants following co-cultivation, which could not be controlled during subsequent sub cultures. Based on these results, a cell density of 0.5 OD600 and a co-cultivation period of 48 h were followed for further transformation studies using Agrobacterium horbouring either of the plasmids. In hygromycin sensitivity test for somatic embryos, it was found that LD50 i.e. necrosis and mortality of 50% of the inoculated embryos; and LD100 i.e. necrosis and mortality of 100% of the inoculated embryos of Crimson Seedless were observed at minimum hygromycin concentrations of 5 and 10 mg l-1, respectively (data not shown).

During initial screening of co-cultured embryos on SM1 containing hygromycin at 5 mg l-1, gradual necrosis of the non-transformed embryos was observed. These necrotic embryos did not show either callusing or repetitive embryogenesis. While, putatively transformed embryos after initial selection on SM1 were transferred on to SM2 containing hygromycin at 10 mg l-1 for final selection. Germination of putatively transformed somatic embryos was observed on WPM supplemented with BA (4.44 µM), IBA (0.49 µM) and hygromycin (10 mg l-1).

Influence of sonication on transformation efficiency

Sonication of explants significantly improved the percentage of embryo survival on selection medium irrespective of the plasmid used for transformation. Duration of sonication coupled with Agrobacterium cell density significantly influenced the number of secondary embryos formed and the transformation efficiency. In general, transformation efficiency increased with increase in sonication period from 1-3 s. In chitinase gene, the transformation efficiency was maximum (9.1%), when somatic embryo were treated with Agrobacterium at 0.5 OD600 and sonicated for 3 s (Table 1). There was a 2.5 fold increase in transformation efficiency by sonication of embryos for 3 s compared to no sonication and treated with Agrobacterium cell density at 0.5 OD600. More or less similar observations were recorded with somatic embryos transformed with Agrobacterium carrying glucanase plasmid. However, the maximum transformation efficiency (6.7%, 3 fold higher than control) with glucanase was recorded with a bacterial cell density of 0.5 OD600 and sonication treatment for 3 s as compared to no sonication control. In the present study, a synergistic effect of bacterial cell density and sonication period on transformation efficiency was observed. This is evident from the fact that there was a 3.5 fold increase in transformation efficiency of somatic embryos sonicated for 3 s with Agrobacterium carrying chitinase plasmid at a cell density of 0.5 OD600 compared to embryos with no sonication but treated with Agrobacterium at 0.2 OD600. While in case of glucanase, the increase in transformation efficiency was 2.3 fold when embryos treated with Agrobacterium at 0.5 OD600 coupled with sonication for 3 s compared to embryos with no sonication but treated with Agrobacterium at 0.2 OD600.

Scanning Electron Microscopic (SEM) view of surface of the embryos after sonication revealed micro-wounding due to sonication that might have allowed the entry of Agrobacterium through and thereby enhanced the infection process. Surface morphology of the control embryos (without sonication) was found to be smooth (Fig. 2A,B) and the embryos sonicated for 1-5 s resulted in limited wounding of the tissue (Fig. 2C). Sonication longer than 5 s resulted in severe tissue damage that subsequently led to embryo mortality (Fig. 2D). In addition, visual observation revealed increased turbidity of Agrobacterium cell suspension due to heavy leaching of cell sap and cell components through micro-wounds in to the solution. The turbidity was more if the sonication was performed longer than 5 s.

Influence of anti-necrotic agents

Addition of anti-oxidants / anti-necrotic agents to the co-cultivation medium significantly improved the number of secondary embryo survival and transformation efficiency irrespective of the plasmid vector used for transformation. In general, silver nitrate, L-cysteine and phenylalanine produced higher responses compared to other anti-necrotic agents. Silver nitrate substantially improved the secondary embryo survival and it gradually increased with the increase in the concentration of silver nitrate (Table 2). At the highest concentration of silver nitrate (5 mg l-1), embryos showed complete mortality with no secondary embryogenesis due to toxicity of silver nitrate on tissues. Among the treatments, phenylalanine (2 mg l-1) affected maximum transformation efficiency (20%), which was on par with silver nitrate (2 mg l-1) with chitinase plasmid. The increase in transformation efficiency in these treatments was 4 fold compared to control (½MS devoid of anti-necrotic agents) (Table 2). More or less similar results were obtained with somatic embryos transformed with Agrobacterium carrying glucanase plasmid. Percentage of embryo survival was higher, when the embryos co-cultured on the medium supplemented with either silver nitrate, trisodium citrate (TSC), sodium thiosulphate or phenylalanine. Among all, sodium thiosulphate (20 mg l-1) or TSC (5 mg l-1) affected a maximum percentage of embryo survival with a transformation efficiency of 25% (Table 2). The increase in transformation efficiency was 7.5 fold compared to control (½MS devoid of anti-necrotic agents).

The putative transformants of chitinase and glucanase induced secondary embryogenesis on ½MS medium supplemented with BA (1 µM) and germination of the transformed embryos was achieved on WPM + BA (4.44 µM) + IBA (0.49 µM) (Fig. 3B,C). Germinated and converted embryos of Crimson Seedless were transferred to plastic cups containing soil-sand-peat (1:1:1) mixture and hardened plants were established in green house (Fig. 3D,E). The integration of anti-fungal genes into the plant genome was confirmed by PCR with gene specific primers (Fig. 4A,B). All putative transformants of Crimson Seedless with chitinase and glucanase selected on SM2 at 12 weeks after co-cultivation produced PCR band sizes corresponding to the respective anti-fungal genes present in plasmids used for transformation. The absence of Agrobacterium contamination in putative transformants was verified by the use of Agrobacterium VirG specific primers (data not shown). Southern blot analysis of the selected transgenic plants showed strong positive signals (compared with positive control) (Fig. 4C) further confirming the integration of T-DNA in to plant genome. Among the two transgenic plants of chitinase analyzed, two showed single copy integration while one line (To1) showed two copies integrated. Whereas, all the three transgenic lines of glucanase had showed single copy integration (Fig. 4D).

Expression of chtinase and ß-1,3-glucanase in transgenic seedlings

The expression of the transgenes was studies in transgenic seedlings by semi quantitative and quantitative RT-PCR. Comparatively a thick band corresponding to chitinase gene was observed in T­01 tranagenic as compared to T­02, while no band was observed in untransformed control (Fig. 5A). Similarly, an uniform band corresponding to ß-1,3-glucanase gene was observed all the three transgenics while no band was observed in untransformed control (Fig. 5B). The relative mRNA expression levels of chitinase gene in transgenic seedlings were 2.3 (T­01) to 1.3 (T­01) (Fig. 5C). Similarly, the relative mRNA expression levels of β-1,3-glucanase gene in transgenic seedlings was ranged from 1.21 (T­03) to 1.45 (T­01) (Fig. 5D).

Activities of chtinase and ß-1,3-glucanase in transgenic seedlings

Activities of chitinase and ß-1,3-glucanase were measured in crude extracts of leaves collected from Southern confirmed transgenic lines showed a substantial increase over untransformed control plants. The line transformed with chitinase and carrying two copies of transgene (To1) showed a maximum (35.7 mU/ g FW) chitinase activity (6 fold increase over control) (Table 3). Lines transformed with glucanase showed a 3-4 fold increase in glucanase activity (Table 4). The increase in activities of chitinase and ß-1,3-glucanase was correlated with the mRNA expression levels of chitinase and glucanase genes.

Fungal tolerance of transgenic plants

The in vitro leaf disc studies conducted to check the tolerance of transgenic lines of grapevine cultivar Crimson Seedless to Downy Mildew fungus showed varied degree of tolerance. There was a 15 to 50% inhibition in hyphal growth compared to control. The transgenic line T01 carrying two copies of chitinase gene showed a maximum of 50% tolerance (expressed as the inhibition of hyphal growth of fungus) as compared to control (Fig. 6). Percentage hyphal inhibition values were lower in case of glucanase transgenics as compared to those of chitinase transgenics. Over all, a linear correlation was observed between recombinant enzymes activity and percentage inhibition of fungal growth.

4. Discussion

In the present study, we demonstrated the suitability of in vitro leaf derived mature somatic embryos of grapevine as target material for Agrobacterium-mediated transformation studies which is in line with earlier studies on grapevine (Perl et al. 1996; Dhekney et al. 2008; Li et al. 2008). Proliferation of transformed somatic embryos occurred via callus or by direct somatic embryogenesis. During direct induction, secondary embryos arose from single epidermal or sub-epidermal cells of the primary embryos (Gray 1995). Such cells represent suitable targets for transformation studies in many plant species including Vitis. Further, it is evident that mature somatic embryos are the most suitable explants for transformation studies in grapevine and other tree species as these multiply rapidly after the transformation event (Christou 1996). After initial screening of co-cultured somatic embryos on SM1 containing 5 mg l-1 hygromycin (LD50) for 4 weeks, embryos were transferred on to SM2 containing 10 mg l-1 hygromycin (LD100) for final selection. This selection method was found appropriate in selecting more number of transformed embryos than selecting directly on higher concentration of hygromycin (10 mg l-1).

Sonication of the somatic embryos significantly improved the transformation efficiency with both the plasmids. In addition, a synergistic effect of bacterial cell density and sonication period on transformation efficiency was observed. This was evident from the results in case of sonication treatments of embryos in Agrobacterium cell density at 0.5 OD600 compared to no sonication but treated with lower cell densities of Agrobacterium. In earlier reports on soybean, sonication of the cotyledons for 2 s prior to co-cultivation significantly increased the transformation efficiency without causing severe tissue damage (Santarem et al. 1998). In another study a 2.2 fold increase in transformation frequency was achieved by sonication of tobacco leaf discs, and the efficiency further increased by 2.5 and 4.1 fold, if sonication was coupled with CaCl2 and acetosyringone treatments, respectively (Kumar et al. 2006). Scanning Electron Microscopic (SEM) view of surface of the embryos after sonication for a period of 1-3 s revealed limited micro-wounding, which might have allowed the entry of Agrobacterium through and thereby enhanced the infection process as observed earlier (Kumar et al. 2006). Our results confirm the earlier findings in soybean cotyledons (Santarem et al. 1998).

Supplementation of anti-necrotic agents in the co-cultivation medium had significant positive influence on the transformation efficiency irrespective of the plasmid used for transformation. Over all, silver nitrate, L-cysteine and phenylalanine produced higher responses compared to other anti-necrotic agents. The increase in transformation with the addition of thiol compounds, L-cysteine, dithiothretol (DTT) and sodium thiosulphate to the co-cultivation medium was attributed to increased T-DNA delivery by thiol inhibition of the activities of Cu and Fe-containing plant pathogen and wound-response enzymes, peroxidases (PODs) and polyphenol oxidases (PPOs), respectively (Olhoft et al. 2001). Thiol inhibited wound- and plant pathogen-induced hypersensitive response renders the plant tissues or cells more susceptible to Agrobacterium infection. As the Agrobacterium infection and tissue necrosis affected, it can be inferred that L-cysteine interacted with tissue response to wound and pathogen infection during co-cultivation, which might have resulted in increased T-DNA delivery (Olhoft and Somers 2001). Earlier, cysteine was believed to increase the frequency of transformation by acting as nutritional supplement during co-cultivation but later realized that it acts through its thiol group. The differential increase in transformation efficiency of the somatic embryos of Crimson Seedless co-cultured in various thiol compounds in the present study might be due to the differential inhibitory action of thiol compounds (Olhoft et al. 2003). Similar to our studies, addition of DTT (1 g l-1) to the post co-cultivation medium significantly improved the stable transformation efficiency of grapevine cv. Thompson Seedless (Li et al. 2009).

Thiol compounds, copper- and iron-chelators were reported to increase the frequency of transformation in soybean earlier (Olhoft et al. 2001). Increased transformation efficiency was reported in immature zygotic embryos of Zea mays L. by the addition of L-cysteine to the solid co-cultivation medium (Frame et al. 2002). As the explant suffers wounding, pathogen infection and environmental stress during co-cultivation, it is expected that a series of wound and pathogen-defense response pathways are active. These defense mechanisms function by releasing phytoalexins and other secondary metabolites which serve as repellants, anti-fungal or anti-bacterial agents and there by induce cell death forming a barrier of dead cells to protect the adjacent healthy tissue (Heath 2000). The increase in the frequency of transformation in the present study is an indicative of a reduced plant defense response to pathogen attack that resulted in reduction in plant cell death. It was also observed in the present study that the addition of anti-oxidants / anti-necrotic agents reduced the tissue necrosis and increased the regeneration of transformants. Further, with the use of anti-necrotic agents in co-cultivation medium the co-cultivation period could be extended to 72 h with no excess bacterial growth. Silver nitrate at lower concentrations acted like a bactericide and suppressed the excessive growth of Agrobacterium on the tissue, which in turn enhanced the overall transformation efficiency confirming the earlier reports in maize (Armstrong and Rout, 2001; Zhao et al., 2001). Further treatment of the explants with anti-necrotic agents might have provided a congenial environment for the interaction of Agrobacterium with the plant cells, thus increasing transformation efficiencies (Enrique-Obregon et al. 1999). The increase in the frequency of transformed somatic embryos resulted by the addition of thiol compounds to the solid co-cultivation medium is independent of binary vectors used for transformation in the present study. Further research into the inhibition of explant wound and pathogen responses may lead to even greater increase in Agrobacterium-mediated transformation of grape and other recalcitrant plant species, especially tree species.

In general, transformed embryos in the present study showed lower degree of somatic embryogenesis, which could be due to hypersensitivity of tissues to Agrobacterium infection (Perl et al. 1996). The regeneration and proliferation processes were reported to be influenced by the transformation process (Bornhoff et al. 2005). Embryo conversion and plantlet growth was also low in transformants compared to non-transformed ones, which could be due the negative influence of antibiotics used for the selection. Grapevine has been reported to be very sensitive to the presence of antibiotic in the medium (Baribault et al. 1990) and the sensitivity depended on the type of the explant. Enhanced activities of chitinase and glucanase were observes in transgenic embryo tissues of Crimson Seedless which had positive correlation with downy mildew tolerance. The increase in the activities of these PR proteins in transgenic plants correlated with their mRNA expression levels assessed by real time PCR. The correlation between resistance and the levels of chitinase and ß-1,3-glucanase activity indicates that these PR proteins may have a role in resistance to the downy mildew fungus confirming the earlier reports by Giannakis et al. (1998). They have reported increased expression of these enzymes in powdery mildew resistant cultivar and their levels increased to substantial amounts upon injury and infection by powdery mildew pathogen. The inhibition of hyphal growth by pure compounds of chitinase and ß-1,3-glucanase from grapevine leaves in the earlier study supports a role of these enzymes in defence of grapevines against downy mildew infection (Giannakis et al. 1998). Further studies on expression and antifungal activity of various isozymes of chitinase and ß-1,3-glucanase in grapevine leaves will be useful for evolving strategies to combat downy and powdery mildew infection.

Conclusion

The efficiency of Agrobacterium-mediated transformation in Crimson Seedless was largely influenced by the co-cultivation period and bacterial cell density used for treating the somatic embryos. Sonication of the somatic embryos for 3 s significantly improved the transformation efficiency of the somatic embryos and prolonged sonication led to severe tissue damage and embryo mortality. Use of anti-oxidants / anti-necrotic agents in co-cultivation medium was found to reduce the tissue necrosis and substantially increased gene transfer and regeneration of transformants. Putatively transformed embryos of the cultivar selected on antibiotic media showed integration of anti-fungal genes as confirmed by PCR and southern blotting. A significant increase in activities of chitinase and glucanase were observed in the leaves of transgenic plants which showed a positive correlation with the degree of fungal tolerance. Studies are underway to screen the transgenic plants of the grapevine cultivar Crimson Seedless overexpressing chitinase and glucanase genes to fungal tolerance under field conditions.

Acknowledgments

Financial support in the form of Senior Research Fellowship (SRF) by the Council of Scientific and Industrial Research (CSIR), Govt. of India to Nookaraju and supply of Chitinase and Glucanase plasmid vectors by Dr. Muthukrishnan Subbarat, Professor in Biochemistry, Kansas State University are gratefully acknowledged.

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