Plant Cell Rep DOI 10.1007/s00299-015-1831-8

ORIGINAL ARTICLE

Agrobacterium-mediated in planta genetic transformation of sugarcane setts Subramanian Mayavan1,2 • Kondeti Subramanyam1,3 • Balusamy Jaganath1 Dorairaj Sathish1 • Markandan Manickavasagam1 • Andy Ganapathi1

•

Received: 14 February 2015 / Revised: 18 June 2015 / Accepted: 23 June 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Key message An efficient, reproducible, and genotypeindependent in planta transformation has been developed for sugarcane using setts as explant. Abstract Traditional Agrobacterium-mediated genetic transformation and in vitro regeneration of sugarcane is a complex and time-consuming process. Development of an efficient Agrobacterium-mediated transformation protocol, which can produce a large number of transgenic plants in short duration is advantageous. Hence, in the present investigation, we developed a tissue culture-independent in planta genetic transformation system for sugarcane using setts collected from 6-month-old sugarcane plants. The sugarcane setts (nodal cuttings) were infected with three Agrobacterium tumefaciens strains harbouring pCAMBIA 1301–bar plasmid, and the transformants were selected against BASTAÒ. Several parameters influencing the in planta transformation such as A. tumefaciens strains, acetosyringone, sonication and exposure to vacuum pressure,

Communicated by P. Lakshmanan. Subramanian Mayavan and Kondeti Subramanyam contributed equally. & Andy Ganapathi [email protected] 1

2

3

Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India Center for Bioenergy, Cooperative Research, Lincoln University of Missouri, Jefferson City, MO 65101, USA Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure links 653, 9000 Ghent, Belgium

have been evaluated. The putatively transformed sugarcane plants were screened by GUS histochemical assay. Sugarcane setts were pricked and sonicated for 6 min and vacuum infiltered for 2 min at 500 mmHg in A. tumefaciens C58C1 suspension containing 100 lM acetosyringone, 0.1 % Silwett L-77 showed the highest transformation efficiency of 29.6 % (with var. Co 62175). The three-stage selection process completely eliminated the chimeric transgenic sugarcane plants. Among the five sugarcane varieties evaluated using the standardized protocol, var. Co 6907 showed the maximum transformation efficiency (32.6 %). The in planta transformation protocol described here is applicable to transfer the economically important genes into different varieties of sugarcane in relatively short time. Keywords Agrobacterium tumefaciens  BASTAÒ  In planta transformation  Sonication  Southern blot hybridization  Sugarcane setts  Vacuum infiltration Abbreviations MS Murashige and Skoog medium hpt II Hygromycin phosphotransferase gus A b-Glucuronidase gene CaMV 35S Cauliflower mosaic virus 35S promoter 35 S poly A 35S Poly A terminator Nos ter Nopaline synthase terminator

Introduction Sugarcane, mainly the complex polyploid interspecific hybrid of Saccharum officinarum L. and S. spontaneum L., is a major food crop in the family Poaceae. It is widely cultivated in tropical and sub-tropical regions for the

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production of sugar, biofuel, electricity and other sugarand fibre-related products (Ming et al. 2006; Lam et al. 2009). Sugarcane is the one of the world’s largest cultivated crop by production quantity and is cultivated in about 26.9 million hectares (FAOSTAT 2013). Brazil is the largest sugarcane producer with 768 TMT, followed by India, China, Thailand, and Pakistan (FAOSTAT 2013). Ever increasing population and increasing sugar consumption forced farmers to extend the cultivation of sugarcane beyond fertile agricultural lands. In the future, the production may not be sufficient to meet the increasing demand for sugar and related products but left with the choice of growing sugarcane in marginal soils with all possible biotic and abiotic production constraints. Hence, it is necessary to develop superior sugarcane cultivars to produce high biomass under unfavourable production conditions. Recent developments in genetic engineering and plant transformation helped researchers to develop sugarcane varieties with improved characters. To date, sugarcane has been transformed by either Agrobacterium tumefaciens or Biolistics methods (Manickavasagam et al. 2004; Zhangsun et al. 2007; Altpeter and Oraby 2010; Joyce et al. 2010; Khamrit et al. 2012). The transformed plants were regenerated using direct, indirect organogenesis or somatic embryogenesis (Arencibia et al. 1998; Manickavasagam et al. 2004; Attia et al. 2005; Lakshmanan et al. 2006; Eldessoky et al. 2011; Taparia et al. 2012). Although these techniques have been successfully employed, the regenerants showed considerable somaclonal variations. In addition, the methods are relatively lengthy and labour-intensive with the potential to cause significant genomic changes. These deficiencies prompted us to develop a sugarcane transformation method without in vitro regeneration. In planta transformation is an alternative method to produce transgenic plants in short time with limited cost and man power. In addition, somaclonal variations could be avoided with the in planta transformation. Ever since Feldmann and Marks (1987) established in planta transformation for Arabidopsis, several researchers have successfully used it for various crops by manipulating a number of parameters, including Agrobacterium strains, target tissue, pre-culture duration, bacterial virulence modifiers and treatments that prime target tissues to Agrobacterium infection (Supartana et al. 2005; Tague and Mantis 2006; Seol et al. 2008; Akbulut et al. 2008; De Oliveira et al. 2009; Li et al. 2009a; Chen et al. 2010; Subramanyam et al. 2013; Mayavan et al. 2013). Agrobacterium-mediated in planta transformation has been successfully established for monocotyledonous plants such as wheat, rice, and maize (Supartana et al. 2006; Lin et al. 2009; Mamontova et al. 2010). Recently, we have developed in planta transformation for sugarcane using seed as an explant (Mayavan et al.

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2013). Even though seeds are good targets for Agrobacterium-mediated in planta transformation, this method is more appropriate to transform homogenous inbred lines but not for commercial sugarcane seeds. This is because, the commercial sugarcane cultivars are mainly complex aneuploid interspecific hybrids and there is no homogeneity in commercial sugarcane seeds. The majority of the sugarcane cultivars currently in cultivation are commercial hybrids and hence, there is a necessity to use alternative explants for in planta transformation. Earlier we have developed a conventional Agrobacterium-mediated transformation method using sugarcane axillary buds as explant and reported good transformation efficiency (Manickavasagam et al. 2004). Development of techniques using mature tissue is important for the improvement of commercial cultivars (Pe´rez-Jime´nez et al. 2013). In addition, sugarcane is a vegetatively propagated crop and stable transformants could be clonally multiplied for distribution to growers (Mordocco et al. 2009). The present investigation was aimed to develop an efficient in planta transformation system for sugarcane using axillary bud as the target tissue. We studied different Agrobacterium strains and different parameters that facilitate Agrobacterium infection and transgene transfer such as the use of acetosyringone, sonication, and vacuum infiltration for developing this technique. Further the standardized method was applied to various sugarcane varieties to evaluate the wider application of this technique in this crop using bar gene, a commercially relevant transgene.

Materials and methods Plant material Five sugarcane hybrid varieties (Co 62175, Co 6304, Co 8021, Co 86032, and Co 6907) were selected for the present study. These sugarcane varieties were cultivated in the research garden, Department of Biotechnology and Genetic Engineering, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Middle portion of stem from 6-month-old healthy sugarcane plants were collected and used for the preparation of target tissue, nodal cuttings called setts (a small portion of the stem with one axillary bud). The setts, approximately 7 cm long, were incubated in 1 % (w/v) bavistin (BASF India Limited, Mumbai, India) solution for 1 h and then rinsed several times with sterile double-distilled water. Minimum inhibitory concentration (MIC) of BASTAÒ The MIC of BASTAÒ [200 g l-1 glufosinate ammonium (Bayer Crop Science, Pinkenba, Australia)] on sett

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sprouting was determined by inoculating the single sugarcane sett in a 500-ml plant tissue culture bottle containing 100 ml of an aqueous solution of BASTAÒ at various concentrations (0, 5, 10, 20, 30, 40, 50 or 60 mg l-1). After inoculation the setts were incubated for 10 days at 28 ± 2 °C under a 16-h photoperiod at a light intensity of 50 lmol m-2 s-1 provided by cool white fluorescent lamps (Philips, Kolkata, India). The particular concentration of BASTAÒ at which the bud present on the sugarcane setts failed to sprout was considered as the MIC and used for selection of transformed sugarcane setts. Fifty sugarcane setts were used per treatment and each treatment was repeated thrice. In another experiment, under greenhouse conditions, 45-day-old sugarcane plants which were raised from wildtype (WT) sugarcane setts were sprayed (50 ml) separately with various concentrations of BASTAÒ (0, 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 g l-1) and the plants were watered on alternate days. The plants were screened after 10 days of BASTAÒ treatment. The particular concentration of BASTAÒ where the plants lost complete chlorophyll was selected as MIC to screen the putatively transformed sugarcane plants under greenhouse conditions. Fifty sugarcane plants were used per treatment and each treatment was repeated thrice. In a similar fashion, the MIC was found for all the sugarcane varieties used in the present study. Agrobacterium strains and plasmid DNA Three different A. tumefaciens strains such as EHA 105, C58C1, and AGL 1 harbouring the binary vector pCAMBIA 1301–bar (Fig. 1) were used in the present investigation. A. tumefaciens EHA 105 is a L,L-succinamopine strain with a C58 chromosomal background and contains pEHA105 as a virulence helper plasmid (Hood et al. 1986, 1993). C58C1 is a nopaline strain with disarmed and a cured version of its plasmid pTiC58 (Van Larebeke et al. 1974; Ashby et al. 1988). AGL 1 is a derivative of C58 containing a deletion in the recA gene that lessens genetic rearrangements of the Ti plasmid in Agrobacterium (Lazo et al. 1991). 35S poly A

LB

The binary vector pCAMBIA 1301–bar was introduced into the above-said three A. tumefaciens strains by tri parental mating using pRK 2013 as a helper vector (Goldberg and Ohman 1984). The T-DNA region of the binary vector contains the hpt II gene expression cassette (CaMV 35S P: hpt II: 35S poly A), bar gene expression cassette (CaMV 35S P: bar: nos ter), and gus A gene expression cassette (CaMV 35S P: gus A: nos poly A) with catalase intron. The Agrobacterium strains were maintained on YEP agar medium supplemented with 50 mg l-1 kanamycin (SRL, Mumbai, India) and 10 mg l-1 rifampicin (SRL, Mumbai, India). In planta transformation of sugarcane setts Agrobacterium suspension preparation A single colony from each of the three Agrobacterium strains was inoculated separately into 10-ml YEP broth amended with 50 mg l-1 kanamycin and 10 mg l-1 rifampicin. The cultures were incubated for 18 h in an orbital shaker with 180 rpm at 28 °C. The Agrobacterium cultures were multiplied by sub-culturing 0.1 volume of bacterial culture into 200-ml YEP broth containing the above-mentioned antibiotics and incubated at 28 °C in an orbital shaker set at 180 rpm until the bacterial broth reached an OD600 of 1.0. The bacterial cells were harvested by centrifuging the culture at 6000 rpm for 8 min. The resulted pellets were suspended in 500 ml of the infiltration medium [liquid MS basal medium (Murashige and Skoog 1962)] containing 5 % sucrose and 0.1 % Silwett L-77 supplemented with various concentrations of acetosyringone (0, 25, 50, 100, 150, and 200 lM) and the optical density was adjusted to an OD600 of 0.6. Agrobacterium infection, co-cultivation, and selection of transformed sugarcane setts and sugarcane plants The sugarcane var. Co 62175 was used to develop the in planta transformation method, and var. Co 6304, Co 8021,

hpt II

CaMV 35S P

CaMV 35S P

Fig. 1 Schematic representation of the binary vector pCAMBIA 1301–bar used in in planta sugarcane setts transformation. The T-DNA region of pCAMBIA 1301–bar showing the assembly of hygromycin expression cassette (CaMV 35S P: hpt II: 35S poly A), bar gene expression cassette (CaMV 35 P: bar: nos ter), and gus A gene expression cassette (CaMV 35S P: gus A: nos poly A). CaMV

nos poly A

Hind III

EcoR I

bar

nos ter

CaMV 35S P

gus A

RB

35S P Cauliflower mosaic virus 35S promoter, hpt II hygromycin phosphotransferase II, 35S poly A Cauliflower mosaic virus 35S poly A terminator, nos ter nopaline synthase terminator, gus A b glucuronidase fusion gene, nos poly A nopaline synthase poly A terminator

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Co 86032, and Co 6907 were used to evaluate the method to a broader genetic background. Several factors influencing the in planta transformation of sugarcane, including A. tumefaciens strains, acetosyringone concentration, duration of sonication, vacuum infiltration pressure, and vacuum duration have been evaluated. To determine the influence of different A. tumefaciens strains on the transformation efficiency, the axillary bud (0.2 cm in length) (Fig. 2a) present on the disinfected sugarcane setts was pricked (5 times, 1 mm in depth) randomly and gently using a sterile hypodermic needle (27G1/1) (Dispovan, New Delhi, India). The setts were then swiftly transferred to different Agrobacterium (EHA 105, C58C1, and AGL 1) suspensions lacking acetosyringone and incubated for 5 h at room temperature. After the incubation, setts were separated from the Agrobacterium suspension and the excess Agrobacterium suspension was removed by air drying on sterile Whatman No. 1 filter paper. The infected sugarcane setts were cocultivated at 25 ± 2 °C for 18 h in a desiccator under complete darkness. In another experiment, the sugarcane setts (pricked) were inoculated into C58C1 Agrobacterium suspension containing various concentrations (0, 25, 50, 100, 150, and 200 lM) of acetosyringone and incubated for 5 h at room temperature. After the incubation, the setts were co-cultivated as described earlier. The influence of sonication duration on the transformation efficiency was evaluated by sonicating the pricked sugarcane setts for various time durations (0, 2, 4, 6, 8 or 10 min) in C58C1 Agrobacterium suspension containing 100 lM acetosyringone using a bath sonicator (model 2510 Branson, Branson Ultrasonics, Kanagawa, Japan). The sonicated sugarcane setts were incubated in the same Agrobacterium suspension for 5 h at room temperature and then co-cultivated as described earlier. To evaluate the influence of vacuum infiltration, the pricked and sonicated (6 min) sugarcane setts were transferred to C58C1 Agrobacterium suspension containing 100 lM acetosyringone and subjected to vacuum infiltration at different pressures (0, 50, 100, 250, 500, and 750 mmHg) for different time durations (0, 1, 2, 3 or 4 min) using a vacuum desiccator (Tarsons, Kolkata, India) connected to a vacuum pump (Indian High Vacuum Pumps, Bangalore, India) (Fig. 2b). After vacuum infiltration, the setts were incubated in the same Agrobacterium suspension for 5 h and then co-cultivated as described earlier. One hundred sugarcane setts were used per treatment and each treatment was repeated thrice. After co-cultivation, the sugarcane setts were washed with sterile double-distilled water containing 500 mg l-1 cefotaxime (Alkem laboratories, Mumbai, India) to kill the Agrobacterium. Each sugarcane sett was transferred to a 500-ml tissue culture bottle containing 100 ml of sterile distilled water with 30 mg l-1 BASTAÒ and incubated at

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25 ± 2 °C under a 16-h photoperiod. During the incubation, the sugarcane setts were partially immersed in the water and the water was replaced with fresh sterile water (containing the same concentration of BASTAÒ and cefotaxime) on alternate days to avoid the bacterial growth. After 25 days, the primary shoot (measuring about 20–25 cm in length) from the 30 mg l-1 BASTAÒ-resistant setts were trimmed, sown in plastic pots (25 9 30 9 20 cm) containing sterile potting mixture (3:1:1 v/v/v garden soil, coir pith and sand). The setts in the plastic pots were irrigated once in 2 days with distilled water containing 30 mg l-1 BASTAÒ. The BASTAÒ-resistant tillers developed from the sugarcane setts were separated from each other and sowed in plastic pots (25 9 30 9 20 cm) containing a sterile potting mixture and allowed to grow for further selection. Forty-five-day-old putatively transformed sugarcane plants (obtained from tillers) were further selected by spraying 1.5 g l-1 BASTAÒ using a hand sprayer and the plants showing a significant resistance to BASTAÒ were chosen for further analysis. GUS histochemical assay Putatively transformed BASTAÒ-resistant young tillers from the sugarcane setts, leaves from 45-day-old BASTAÒ (1.5 g l-1)-resistant sugarcane plants, and the respective non-transformed (NT) controls were assessed for gus A gene expression by GUS histochemical assay as described by Jefferson et al. (1987). The aforesaid plant materials were incubated overnight at 37 °C in 2 mM X-Gluc (5Bromo-4-chloro-3-indolyl b-D-glucoronide) in a phosphate buffer (pH 7.0) containing 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1 % v/v Triton X-100. The chlorophyll was removed using 95 % ethanol after X-Gluc staining. Molecular analysis of putatively transformed sugarcane plants Genomic DNA isolation and polymerase chain reaction (PCR) The genomic DNA was isolated from 2-month-old GUSpositive putatively transformed and NT sugarcane plants by following the method described by Dellaporta et al. (1983). To verify the presence of the bar gene in putatively transformed sugarcane plants, PCR was performed with the aid of bar gene-specific primers (barFP: 50 -ATCGTCAACCACTACATCGAGAC-30 ; barRP: 50 0 CCAGCTGCCAGAAACCCACGTC-3 ) which specifically amplify 462 bp coding region of the bar gene. The PCR was carried out in a PTC-100TM thermal cycler (MJ

Plant Cell Rep

Fig. 2 Agrobacterium tumefaciens-mediated in planta genetic transformation of sugarcane setts and production of transformed sugarcane plants var. Co 6907. a sugarcane setts collected from 6-month-old field-grown sugarcane plants and used for Agrobacterium infection; b vacuum infiltration of sonicated sugarcane setts in Agrobacterium suspension using vacuum pump and desiccator; c sugarcane sett sprouting from the axillary bud after 3 days of incubation in distilled water containing 30 mg l-1 BASTAÒ; d non-transformed (NT)

sugarcane sett after 3 days of incubation in distilled water containing 30 mg l-1 BASTAÒ; e, f primary shoot development from the axillary bud present on the putatively transformed sugarcane sett after 7 and 10 days, respectively, in distilled water containing 30 mg l-1 BASTAÒ; g–i primary shoot development from the axillary bud present on the putatively transformed sugarcane sett after 15, 20, and 25 days, respectively, in distilled water containing 30 mg l-1 BASTAÒ

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research Inc, Waltham, MA, USA) programmed with an initial denaturation of DNA at 95 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 61 °C for 1 min and 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. The binary vector pCAMBIA 1301–bar and NT sugarcane plant genomic DNA was used as positive and negative control, respectively. The amplified products were analyzed by electrophoresis on a 1 % (w/v) agarose gel. Southern blot hybridization For Southern blot hybridization, 10 lg of genomic DNA from each of the PCR-positive and NT sugarcane plants, and 5 lg of binary vector pCAMBIA 1301–bar was digested overnight with EcoR I at 37 °C. The digested samples were resolved by electrophoresis on a 1 % agarose gel, and the size-fractioned DNA fragments were blotted onto a Hybond N? nylon membrane (GE healthcare, Buckinghamshire, UK) as essentially described by Sambrook et al. (1989). The blot was hybridized at 55 °C with alkaline phosphatase (ALP)-labelled 462 bp PCR-amplified and purified product of bar gene for 8 h. The hybridized membrane was washed with primary and secondary wash buffers according to the manufacturer’s instructions (GE healthcare, Buckinghamshire, UK), subjected to chemiluminescent development using CDP Star substrate (GE healthcare, Buckinghamshire, UK), and exposed to X-ray film (Kodak, Mumbai, India). The binary vector pCAMBIA 1301–bar and NT sugarcane plant genomic DNA was used as positive and negative control, respectively. Influence of genotype on in planta transformation After determining the various parameters influencing the in planta transformation of sugarcane setts using var. Co 62175, the developed protocol was evaluated with four other varieties, Co 6304, Co 8021, Co 86032, and Co 6907. Statistical analysis Data were analyzed using one-way ANOVA, and the differences contrasted using Duncan’s multiple range tests (DMRT). All statistical analyses were performed at the level of P \ 0.05 using SPSS 10.0 (SPSS Inc. USA). Results and discussion Sugarcane is amenable to Agrobacterium-mediated genetic transformation. However, the transformed sugarcane plants were recovered through in vitro regeneration, which requires extensive tissue culture manipulations stretching several months. In addition, it is also associated with the

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risk of somaclonal variations (Joyce et al. 2014). Hence, we developed an efficient in planta transformation system for sugarcane using sugarcane setts as explant.

MIC of BASTAÒ Chimerism is a serious problem and it does occur in Agrobacterium-mediated genetic transformation. Hence, avoiding chimerism is an important aspect of transgenic plant production. The inclusion of selection markers in the T-DNA region of the binary vector is an effective strategy to eliminate chimeric cells/tissues and allow only transformed cell/tissue to proliferate. The herbicide-resistant plant selection marker—the bar gene—which confers resistance to BASTAÒ was used in the present study. The BASTAÒ was used as a selection agent in a number of monocotyledonous plants such as maize, rice, wheat, and sugarcane (Fromm et al. 1990; Christou et al. 1991; Vasil et al. 1992; Chowdhury and Vasil 1992; Enrı´quez-Obrego´n et al. 1998; Manickavasagam et al. 2004; Mayavan et al. 2013). In the present study, the sugarcane setts collected from 6-month-old field-grown plants and 45-day-old sugarcane plants raised from the setts were treated with various concentrations of BASTAÒ. Among the various concentrations tested, BASTAÒ from 30 mg l-1 rate prevented sprouting of sugarcane setts and the plants (45-dayold) sprayed with 1.5 g l-1 BASTAÒ showed necrotic symptoms and died. Hence, 30 mg l-1 and 1.5 g l-1 BASTAÒ was assigned as MIC to select the transformed sugarcane setts and 45-day-old sugarcane plants, respectively. MIC was found to be similar for all the varieties used for the in planta genetic transformation (data not shown). Mayavan et al. (2013) selected the transformed sugarcane seedlings by germinating in the sterile MS liquid medium supplemented with 2.0 mg l-1 BASTAÒ and then 2-month-old plants by spraying with 2.0 g l-1 BASTAÒ. In a similar fashion, the transformed Arabidopsis, Medicago truncatula, Brassica rapa, and Jatropha curcas were selected by spraying BASTAÒ (Mengiste et al. 1997; Trieu et al. 2000; Qing et al. 2000; Jaganath et al. 2014). Influence of different Agrobacterium strains and acetosyringone on transformation efficiency It is a well-known fact that the transformation efficiency depends on the ability of Agrobacterium to infect the explant. In the current investigation, three A. tumefaciens (EHA 105, C58C1, and AGL1) strains were used to infect the sugarcane setts of var. Co 62175 in the absence of acetosyringone. Among these, A. tumefaciens C58C1 showed the highest transformation efficiency. About 25.6 % of sugarcane setts infected with A. tumefaciens

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C58C1, sprouted in 30 mg l-1 BASTAÒ solution and 33.6 % of them showed significant resistance to 1.5 g l-1 BASTAÒ. Nearly 73.2 % of the BASTA-resistant plants expressed the gus A gene. The overall transformation efficiency with C58C1 strain was 6.3 % (Table 1). The A. tumefaciens strains EHA 105 and AGL 1 showed 4.6 and 3.6 % of transformation efficiency, respectively. The Agrobacterium strains EHA 105, C58C1, and AGL 1 not only have a different chromosomal background, but also different vir helper plasmid with different levels of activating potency. It was expected for these reasons the C58C1 had stronger capability to infect the sugarcane setts than EHA 105 and AGL 1. Hence, in the present study, A. tumefaciens strain C58C1 was used to evaluate the other parameters influencing the in planta transformation. The A. tumefaciens strain C58C1 was successfully used for the genetic transformation of carrot, Arabidopsis, and wheat (Pawlicki et al. 1992; Wilson et al. 1996; Cheng et al. 1997; Zale et al. 2009). The inclusion of acetosyringone dramatically enhanced the transformation efficiency (Table 2). In the presence of 100 lM acetosyringone, 32.6 % of Agrobacterium-infected setts were sprouted in 30 mg l-1 BASTAÒ solution, reaching a transformation efficiency of 11.6 % (Table 2). A similar improvement of transformation with acetosyringone was reported in switchgrass, rice, Medicago truncatula, Arabidopsis, and cowpea (Chen et al. 2010; Naseri et al. 2012; Trieu et al. 2000; Li et al. 2009b; Adesoye et al. 2010). Application of acetosyringone, above 100 lM reduced sett sprouting ability, which ultimately led to a lower transformation efficiency (Table 2). This could be due to the presence of alcohol as solvent to dissolve the acetosyringone (Subramaniam et al. 2009).

Influence of sonication and vacuum infiltration on transformation efficiency The cells present in the meristematic region play an important role in developing transgenic plants. Since, the meristematic cells are present deep inside the tissue, there will be a less chance of Agrobacterium infection, which led to low transformation efficiency. This problem could be overcome by creating microcavities across the explant by means of sonication. Sonication was successfully employed to improve the genetic transformation efficiency of several monocotyledon and dicotyledonous plants (Solı´s et al. 2003). In the present investigation, the percentage of infected setts sprouting, and the number of GUS-positive plants were gradually increased with the increasing duration of sonication, with 6 min being the optimum duration where 53 % of infected sugarcane setts sprouted in 30 mg l-1 BASTAÒ solution and 71 % of them showed significant resistance to 1.5 g l-1 BASTAÒ. Nearly 57.4 % of the BASTA-resistant plants expressed the gus A gene with a transformation efficiency of 21.6 % (Table 3). Longer duration of sonication has an inhibitory effect on cells, such as cell lysis and suppression of RNA and protein synthesis (Joersbo and Brunstedt 1992). Chen et al. (2010) reported 31.3 % transformation efficiency in Panicum virgatum by sonicating 3-day-old seedlings for 1 min. Mayavan et al. (2013) applied 10 min of sonication to transform sugarcane seeds, which produced 32.4 % of transformation efficiency. Sonication has been successfully applied in Cicer arietinum, Vigna unguiculata, Banana, Catharanthus roseus and eggplant (Indurker et al. 2010; Bakshi et al. 2011; Subramanyam et al. 2011; Wang et al. 2012; Subramanyam et al. 2013).

Table 1 Influence of Agrobacterium strains on in planta transformation efficiency of sugarcane var. Co 62175 Serial number

Agrobacterium strain

No. of setts infected

Mean no. of setts sproutedA

Mean no. of plants survived after BASTA sprayB

Mean no. of GUS? plantsC

Transformation efficiency (%)D

1

EHA 105

100

20.3 ± 0.2b

6.6 ± 0.1b

4.6 ± 0.1b

4.6 ± 0.1b

100

a

8.6 ± 0.2

a

a

6.3 ± 0.1a

5.3 ± 0.1

c

c

3.6 ± 0.1c

2 3

C58C1 AGL 1

100

25.6 ± 0.2

c

18.0 ± 0.1

6.3 ± 0.1 3.6 ± 0.1

The bud present on the sugarcane setts was pricked randomly and infected for 5 h with different A. tumefaciens strains harbouring pCAMBIA 1301–bar plasmid suspension containing 5 % sucrose and 0.1 % Silwett L-77 (without acetosyringone) and co-cultivated for 18 h in a desiccator. One hundred sugarcane setts were used per treatment and each treatment was repeated thrice. Mean values of three independent experiments (±) with standard errors. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5 % level A

The infected sugarcane setts were sprouted in a tissue culture bottle containing 100 ml of sterile distilled water with 30 mg l-1 BASTAÒ

B

The putatively transformed sugarcane plants were sprayed with 1.5 g l-1 BASTAÒ and the results were recorded after 10 days

C

No. of BASTAÒ (1.5 g l-1)-resistant sugarcane plants showing gus A gene expression

D

Transformation efficiency = no. of GUS? plants/total no. of sugarcane setts infected 9 100

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Plant Cell Rep Table 2 Influence of different concentrations of acetosyringone on in planta transformation efficiency of sugarcane var. Co 62175 Serial number

Acetosyringone concentration (lM)

1

0

2

25

3

50

4 5 6

25.6 ± 0.3f

8.6 ± 0.2e

6.3 ± 0.2f

6.3 ± 0.2f

100

e

15.3 ± 0.3

d

7.6 ± 0.2

e

7.6 ± 0.2e

17.6 ± 0.2

b

9.0 ± 0.3

d

9.0 ± 0.3d

19.3 ± 0.3

a

11.6 ± 0.2

a

11.6 ± 0.2a

17.3 ± 0.2

b

10.3 ± 0.4

b

10.3 ± 0.4b

16.0 ± 0.2

c

9.6 ± 0.3

c

9.6 ± 0.3c

100

200

Transformation efficiency (%)D

100

100

150

Mean no. of GUS? plantsC

Mean no. of setts sproutedA

100

100

Mean no. of plants survived after BASTA sprayB

No. of setts infected

100

26.3 ± 0.4

c

29.6 ± 0.3

a

32.6 ± 0.2

b

30.0 ± 0.3

d

28.3 ± 0.3

The bud present on the sugarcane setts was pricked randomly and infected for 5 h with A. tumefaciens strain C58C1 harbouring pCAMBIA 1301–bar plasmid suspension containing 5 % sucrose, 0.1 % Silwett L-77, and different concentrations of acetosyringone. After 5 h of infection, the sugarcane setts were co-cultivated for 18 h in a desiccator. One hundred sugarcane setts were used per treatment and each treatment was repeated thrice. Mean values of three independent experiments (±) with standard errors. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5 % level A

The infected sugarcane setts were sprouted in a tissue culture bottle containing 100 ml of sterile distilled water with 30 mg l-1 BASTAÒ

B

The putatively transformed sugarcane plants were sprayed with 1.5 g l-1 BASTAÒ and the results were recorded after 10 days

C

No. of BASTAÒ (1.5 g l-1)-resistant sugarcane plants showing gus A gene expression

D

Transformation efficiency = no. of GUS? plants/total no. of sugarcane setts infected 9 100

Table 3 Influence of different sonication durations on in planta transformation efficiency of sugarcane var. Co 62175 Serial number

Sonication duration (min)

1

0

2

2

3

4

No. of setts infected

Mean no. of setts sproutedA

Mean no. of plants survived after BASTA sprayB

Mean no. of GUS? plantsC

Transformation efficiency (%)D

100

32.6 ± 0.4f

19.3 ± 0.2f

11.6 ± 0.2f

11.6 ± 0.2f

100

34.3 ± 0.4

e

e

15.6 ± 0.2

e

15.6 ± 0.2e

42.3 ± 0.2

c

18.3 ± 0.3

c

18.3 ± 0.3c

a

37.6 ± 0.4

21.6 ± 0.2

a

21.6 ± 0.2a

100

26.6 ± 0.3

c

31.3 ± 0.2

a

4

6

100

53.0 ± 0.3

5

8

100

45.6 ± 0.2b

33.3 ± 0.2b

19.6 ± 0.2b

19.6 ± 0.2b

100

d

d

d

16.3 ± 0.2d

6

10

38.3 ± 0.3

28.6 ± 0.2

16.3 ± 0.2

The bud present on the sugarcane setts was pricked randomly and sonicated for different time durations in A. tumefaciens strain C58C1 harbouring pCAMBIA 1301–bar plasmid suspension containing 5 % sucrose, 0.1 % Silwett L-77, and 100 lm of acetosyringone. After 5 h of infection, the sugarcane setts were co-cultivated for 18 h in a desiccator. One hundred sugarcane setts were used per treatment and each treatment was repeated thrice. Mean values of three independent experiments (±) with standard errors. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5 % level A

The infected sugarcane setts were sprouted in a tissue culture bottle containing 100 ml of sterile distilled water with 30 mg l-1 BASTAÒ

B

The putatively transformed sugarcane plants were sprayed with 1.5 g l-1 BASTAÒ and the results were recorded after 10 days

C

No. of BASTAÒ (1.5 g l-1)-resistant sugarcane plants showing gus A gene expression

D

Transformation efficiency = no. of GUS? plants/total no. of sugarcane setts infected 9 100

Though the sonication creates the microcavities across the bud present on the setts, it may be more effective to direct the Agrobacterium to the meristematic region through the cavities to increase transformation efficiency. This can be achieved by vacuum infiltration. The application of vacuum may cause gases to withdraw from the interior parts of buds through stomata and probably through wounding sites. As the vacuum is broken and pressure rapidly increases, the Agrobacterium suspension may be driven into the bud to replace these gases. This phenomenon may expose Agrobacterium to plant cells that are most susceptible to transformation than those present on

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the bud epidermis. In the present investigation, the sonicated sugarcane setts were vacuum infiltered in Agrobacterium suspension at different vacuum pressures for different time durations (Table 4). The percentage of sugarcane setts sprouting in 30 mg l-1 BASTAÒ solution and vacuum pressure at 500 mmHg was found to be optimum for sugarcane setts infection. When the 6-min-sonicated sugarcane setts were vacuum infiltered for 2 min at 500 mmHg, 69.3 % of Agrobacterium-infected setts were sprouted in 30 mg l-1 BASTAÒ solution and of these 71.1 % of plants showed significant resistance to 1.5 g l-1 BASTAÒ. About 60 % of the BASTA-resistant plants

Plant Cell Rep Table 4 Influence of different vacuum pressures and vacuum durations on in planta transformation efficiency of sugarcane var. Co 62175 Serial number 1

Vacuum infiltration pressure (mmHg) 0

2

50

Vacuum infiltration duration (min)

No. of setts infected

Mean no. of setts sproutedA

Mean no. of plants survived after BASTA sprayB

Mean no. of GUS? plantsC

Transformation efficiency (%)D

0

100

53.0 ± 0.2m

37.6 ± 0.3m

21.6 ± 0.2ga

21.6 ± 0.2ga

1

100

56.3 ± 0.3

l

39.6 ± 0.2

23.6 ± 0.3

23.6 ± 0.3f

da

50 50

2 3

100 100

59.6 ± 0.2 65.3 ± 0.3d

42.0 ± 0.2 46.3 ± 0.4da

25.0 ± 0.3 27.6 ± 0.2b

25.0 ± 0.3da 27.6 ± 0.2b

5

50

4

100

62.0 ± 0.2fb

43.6 ± 0.3g

26.3 ± 0.2c

26.3 ± 0.2c

41.3 ± 0.4

j

23.6 ± 0.3

f

23.6 ± 0.3f

43.6 ± 0.3

g

24.6 ± 0.2

e

24.6 ± 0.2e

47.3 ± 0.4

c

26.3 ± 0.1

c

26.3 ± 0.1c

100

7

100

1 2

100 100

58.0 ± 0.4

j

61.3 ± 0.4

g c

ib

f

3 4 6

i

l

8

100

3

100

66.3 ± 0.2

9

100

4

100

62.6 ± 0.3f

44.6 ± 0.3f

100

60.6 ± 0.4

h

42.6 ± 0.2

i

63.3 ± 0.2

e

44.3 ± 0.3

fa

68.6 ± 0.3

b

48.0 ± 0.2

b

65.3 ± 0.4

d

45.6 ± 0.4

e

e

45.3 ± 0.3

ea

10 11 12 13

250 250 250 250

1 2 3 4

100 100 100

14

500

1

100

63.6 ± 0.3

15

500

2

100

69.3 ± 0.2a

16 17

500 500

3 4

100 100

66.0 ± 0.4

ca

62.3 ± 0.4

fa

24.0 ± 0.2ea

24.6 ± 0.3

e

24.6 ± 0.3e

25.6 ± 0.4

d

25.6 ± 0.4d

27.3 ± 0.4

ba

27.3 ± 0.4ba

26.0 ± 0.2

ca

26.0 ± 0.2ca

26.3 ± 0.3

c

26.3 ± 0.3c

49.3 ± 0.4a

29.6 ± 0.2a

29.6 ± 0.2a

46.6 ± 0.3

d

27.6 ± 0.2

b

27.6 ± 0.2b

42.3 ± 0.2

ia

25.0 ± 0.1

da

25.0 ± 0.1da

e

18 19

750 750

1 2

100 100

60.6 ± 0.3 57.3 ± 0.2k

43.0 ± 0.2 40.6 ± 0.3k

24.6 ± 0.2 23.3 ± 0.1fa

24.6 ± 0.2e 23.3 ± 0.1fa

20

750

3

100

52.0 ± 0.3n

36.6 ± 0.2n

21.0 ± 0.3g

21.0 ± 0.3g

100

o

o

h

19.3 ± 0.2h

21

750

4

48.3 ± 0.2

h

24.0 ± 0.2ea

h

33.3 ± 0.2

19.3 ± 0.2

The bud present on the sugarcane setts was pricked randomly, sonicated for 6 min in A. tumefaciens strain C58C1 harbouring pCAMBIA 1301– bar plasmid suspension containing 5 % sucrose, 0.1 % Silwett L-77, and 100 lm of acetosyringone and vacuum infiltered at different vacuum pressures for different time durations in Agrobacterium suspension. After 5 h of infection, the sugarcane setts were co-cultivated for 18 h in a desiccator. One hundred sugarcane setts were used per treatment and each treatment was repeated thrice. Mean values of three independent experiments (±) with standard errors. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5 % level A

The infected sugarcane setts were sprouted in a tissue culture bottle containing 100 ml of sterile distilled water with 30 mg l-1 BASTAÒ

B

The putatively transformed sugarcane plants were sprayed with 1.5 g l-1 BASTAÒ and the results were recorded after 10 days No. of BASTAÒ (1.5 g l-1)-resistant sugarcane plants showing gus A gene expression

C D

Transformation efficiency = no. of GUS? plants/total no. of sugarcane setts infected 9 100

expressed the gus A gene with a transformation efficiency of 29.6 % (Table 4). A 3-min vacuum infiltration was found to be optimum at 50, 100, and 250 mmHg, wherein 65.3, 66.3, and 68.6 % of infected setts sprouted in the presence of 30 mg l-1 BASTAÒ and showed 27.6, 26.3, and 27.3 % of transformation efficiency, respectively (Table 4). On the other hand, 1-min vacuum infiltration was found to be optimum at 750 mmHg, wherein 60.6 % of infected setts was sprouted in 30 mg l-1 BASTAÒ solution and showed 24.6 % of transformation efficiency (Table 4). Based on these data, 6-min sonication followed by 2-min vacuum infiltration at 500 mmHg in Agrobacterium C58C1 suspension supplemented with 100 lM acetosyringone and 18 h co-cultivation were found to be optimum to achieve maximum transformation efficiency of 29.6 % (with var. Co 62175). This result is similar to what

was reported in citrus, banana, cowpea, lentil, sugarcane, and brinjal (De Oliveira et al. 2009; Subramanyam et al. 2011; Bakshi et al. 2011; Chopra and Apartna 2012; Mayavan et al. 2013; Subramanyam et al. 2013). Selection of transformed sugarcane plants Selection agent and selection pressure are the two important parameters that prevent or minimize the development of chimeras or escapes in the in planta transformation. In the present study, we have been applying selection pressure at three developmental stages—initially at the stage of setts sprouting, then during tiller’s formation, and finally at the greenhouse stage where plants were grown for 45 days. The co-cultivated sugarcane setts were washed with 500 mg l-1 cefotaxime and then grown in the presence of

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Plant Cell Rep

Fig. 3 Screening of putatively transformed sugarcane plants by BASTAÒ spray assay and GUS histochemical analysis. a putatively transformed sugarcane tillers developed from 30 mg l-1 BASTAÒresistant sugarcane sett; b forty-five-day-old putatively transformed sugarcane plants showing healthy leaves after 1 week of BASTAÒ spray (1.5 g l-1) in the greenhouse; c forty-five-day-old non-

transformed (NT) sugarcane plant showing dried leaves without chlorophyll after 1 week of BASTAÒ spray (1.5 g l-1) in the greenhouse; d the putatively transformed tillers showing blue colour when stained with X-gluc; e the NT tillers showing no blue colour following X-gluc staining

30 mg l-1 BASTAÒ. The transformed buds were sprouted (Fig. 2c) after 3 days of BASTAÒ inoculation and developed into primary shoots, which showed significant resistance to 30 mg l-1 BASTAÒ (Fig. 2e–i). The nontransformed (NT) buds treated with BASTAÒ failed to respond (Fig. 2d). The transgenic shoots were trimmed and transferred to plastic pots containing a sterile potting mixture for the second-stage selection against 30 mg l-1 BASTAÒ. The tillers developed (Fig. 3a) were separated from the setts and grown in plastic pots containing the potting mixture for further selection in the greenhouse. Forty-five-day-old putatively transformed greenhousegrown plants were sprayed with 1.5 g l-1 BASTAÒ. After 1 week of BASTAÒ spray, the transformed sugarcane plants showed significant resistance against 1.5 g l-1 of BASTAÒ and grew further (Fig. 3b). Whereas, non-transgenic plants showed growth retardation and loss of chlorophyll and they finally perished (Fig. 3c). The BASTAÒ-resistant plants were selected for further analysis.

Screening of transformed sugarcane plants

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GUS histochemical analysis Histochemical analysis of gus A gene expression was used to analyze the effect of various parameters, including Agrobacterium strain, acetosyringone, sonication, vacuum pressure, and vacuum infiltration duration on the transformation efficiency. An intense blue colour was observed in the putatively transformed tillers (Fig. 3d) and leaf when stained with X-gluc indicating gus A expression. On the other hand, the tillers developed from non-transgenic (NT) sugarcane setts (Fig. 3e) failed to show blue colour after X-gluc staining. Molecular confirmation of transformed sugarcane plants The putatively transformed 2-month-old sugarcane plants were analyzed for the bar gene integration by performing PCR on GUS-positive lines. The genomic DNA from putatively transformed and NT sugarcane plant leaves was

Plant Cell Rep

Fig. 4 Detection of bar gene integration in putatively transformed sugarcane plants genome. a PCR amplification of the bar gene from the genomic DNA of putatively transformed sugarcane plants. Lane 1 100 bp plus DNA ladder; Lane 2 pCAMBIA 1301–bar as a positive control; Lanes 3–7 putatively transformed sugarcane plants genomic DNA carrying bar gene; Lane 8 non-transformed (NT) sugarcane

genomic DNA as a negative control. b Southern blot analysis of putatively transformed sugarcane plants. Lane 1 pCAMBIA 1301–bar as a positive control; Lanes 2–6 putatively transformed sugarcane genomic DNA samples; Lane 7 NT sugarcane plant genomic DNA. DNA samples were digested with EcoR I restriction enzyme and PCR-amplified product of the bar was used as a probe

Table 5 The integration pattern (copy number) of bar gene into the transformed sugarcane genome across the 5 sugarcane varieties revealed by Southern blot hybridization Serial number

Sugarcane variety

No. of transformed plants showing single copy of bar gene

No. of transformed plants showing two copies of bar gene

No. of transformed plants showing three copies of bar gene

Total no. of transformed plants

1

Co 62175

7

10

12

29

2

Co 6304

5

9

14

28

3

Co 8021

8

6

16

30

4

Co 86032

7

8

12

27

5

Co 6907

9

8

15

32

isolated by following the method described by Dellaporta et al. (1983). In PCR, the bar gene-specific primers amplified 462 bp coding region of the bar gene from pCAMBIA 1301–bar plasmid (Fig. 4a, lane 2) and putatively transformed (Fig. 4a, lanes 3–7) sugarcane plants. It indicates the integration of bar gene in the sugarcane genome. On the other hand, NT sugarcane plant genomic DNA showed no amplification with bar gene-specific primers (Fig. 4a, lane 8). Southern blot analysis revealed the copy number of bar gene in sugarcane genome and independent transgenic events. Genomic DNA was digested with EcoR I, which recognizes a single site within the T-DNA region of the pCAMBIA 1301–bar vector and hybridized with the probe prepared using PCR-amplified product of bar gene. DNA from NT plants was used as a negative control and showed no hybridization (Fig. 4b, lane 7), whereas, the binary vector pCAMBIA 1301–bar generated hybridization signal

(Fig. 4b, lane 1). The T-DNA of the pCAMBIA 1301–bar has only one EcoR I site; located between the hpt II and bar genes. Hence, probing with bar gene sequence gives an indication of the number of bar gene copies integrated. Transformed sugarcane plants showed up to three copies of T-DNA integration (Fig. 4b, lanes 2–6), and the hybridization patterns were non-identical due to different transformation events. The integration patterns (copy number) of bar gene in the transformed sugarcane genome across the five varieties are given in Table 5. Influence of genotype on in planta transformation To evaluate the amenability of in planta transformation developed using sugarcane var. Co 62175, the same protocol was adopted to screen another four varieties such as Co 6304, Co 8021, Co 86032, and Co 6907. Among the five varieties evaluated, Co 6907 was found to be the best

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Plant Cell Rep Table 6 Influence of genotype on in planta sugarcane transformation Serial number

Sugarcane variety

No. of setts infected

Mean no. of setts sproutedA

Mean no. of plants survived after BASTA sprayB

Mean no. of GUS? plantsC

Transformation efficiency (%)D

1

Co 62175

100

69.3 ± 0.2c

49.3 ± 0.4c

29.6 ± 0.2c

29.6 ± 0.2c

100

66.3 ± 0.2

d

d

28.3 ± 0.1

d

28.3 ± 0.1d

70.3 ± 0.4

b

30.3 ± 0.3

b

30.3 ± 0.3b

62.6 ± 0.1

e

27.0 ± 0.2

e

27.0 ± 0.2e

73.6 ± 0.2

a

32.6 ± 0.2

a

32.6 ± 0.2a

2

Co 6304

3

Co 8021

4

Co 86032

5

Co 6907

100 100 100

47.6 ± 0.4

b

52.3 ± 0.3

e

44.6 ± 0.2

a

54.6 ± 0.2

The bud present on the sugarcane setts was pricked randomly, sonicated for 6 min in A. tumefaciens strain C58C1 harbouring pCAMBIA 1301– bar plasmid suspension containing 5 % sucrose, 0.1 % Silwett L-77, and 100 lm of acetosyringone and vacuum infiltered at 500 mmHg for 2 min in Agrobacterium suspension. After 5 h of infection, the sugarcane setts were co-cultivated for 18 h in a desiccator. One hundred sugarcane setts were used per treatment and each treatment was repeated thrice. Mean values of three independent experiments (±) with standard errors. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5 % level A

The infected sugarcane setts were sprouted in a tissue culture bottle containing 100 ml of sterile distilled water with 30 mg l-1 BASTAÒ

B

The putatively transformed sugarcane plants were sprayed with 1.5 g l-1 BASTAÒ and the results were recorded after 10 days

C

No. of BASTAÒ (1.5 g l-1)-resistant sugarcane plants showing gus A gene expression

D

Transformation efficiency = no. of GUS? plants/total no. of sugarcane setts infected 9 100

responding one with a transformation efficiency of 32.6 %, followed by Co 8021, Co 62175, Co 6304, and Co86032 (Table 6). This indicates that the in planta transformation protocol developed here could be useful to transform different varieties/cultivars of sugarcane using setts as explant.

Acknowledgments The authors are grateful to University Grants Commission (UGC), Government of India, for the financial support [No.F.31-239/2005 (SR)] to carry out the present work. The corresponding author is thankful to University Grants Commission (UGC), Govt. of. India for providing Fellowship under UGC–BSR scheme. All the authors are thankful to Prof. A.S Rao, Department of Biotechnology & Genetic Engineering, Bharathidasan University, Tiruchirappalli for his valuable suggestions in improving the manuscript.

Conclusion

Conflict of interest of interest.

In conclusion, an efficient in planta Agrobacterium-mediated genetic transformation was developed for sugarcane using setts as explant. When the bud was pricked with a fine needle, sonicated for 6 min in A. tumefaciens strain C58C1 harbouring pCAMBIA 1301–bar plasmid suspension containing 5 % sucrose, 0.1 % Silwett L-77, and 100 lm of acetosyringone, and vacuum infiltered at 500 mmHg for 2 min in Agrobacterium suspension recorded a maximum transformation efficiency of 29.6 % (with var. Co 62175). When we applied the same protocol to another 4 varieties, var. 6907 was emerged as a best responding variety with a transformation efficiency of 32.6 %. This is the first report on in planta Agrobacteriummediated genetic transformation of sugarcane using setts as explant. The in planta transformation protocol developed in the present investigation would be useful for the genetic improvement of sugarcane. Author contribution statement AG and MM conceived and designed the experiments. SM and KS performed the experiments. KS and BJ performed the Southern hybridization experiments. BJ and DS analyzed the data. KS and AG contributed to the writing of the manuscript.

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The authors declare that they have no conflict

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