IN-PLANTA TRANSFORMATION METHOD MEDIATED WITH Agrobacterium tumefaciens FOR T-DNA TRANSFER IN TABLE GRAPE ( Vitis vinifera L . )

The aim of the research is to investigate a simple method of in planta transformation method for T-DNA transfer in table grape. The T-DNA harbored the S0SPS1 gene under the control of promoter of the 35S CaMV from the Cauliflower Mosaic Virus and contained the NPTII gene, a kanamycin-resistant gene as a selectable marker for transformant selection. Six-month plants originated from cuttings were used as target plants. We explored two methods of in planta transformation, namely ”dipping” and “sweeping”. For both methods, the leaves of the target plants were removed and those of shoots without leaves were used as the target of transformation. In the “dipping method”, those shoots were dipped with the agrobacterial suspension for 60 seconds. However, for the “sweeping method”, the scars (the spots where leaves were removed) were swept with agrobacterial suspension using a cotton bud. Those treated non-leafy-shoots (from both methods) then were grown to be leafy shoots. Those leafy shoots then were cut and transplanted into the soil and grown to be a whole plant. The leaves of those plants then were taken as samples for DNA extraction and PCR using primers of NPTII gene (Forward: 5’-GTCATCTCACCTTCCTCCTGCC-3’; Reverse: GTCGCTTGGTCGGTCATTTCG-3’) with expected amplified band of 550 bp. We found that only the “sweeping method” plants amplified the 550 bp bands, while those of the “dipping method” did not. We suggest that the T-DNA was successfully integrated into the genome of plants treated with the “sweeping method” but not with the “dipping method”. Leaf sugar content ( o Brix) of PCR-positive vines was higher than those of the wild-type vines, ensuring the integration of the T-DNA into the plant genome.


INTRODUCTION
Genetic transformation is an efficient way to improve the characters of plants. The most common methods for the introduction of DNA into plant cells use Agrobacterium tumefaciens or rapidly propelled tungsten microprojectiles that have been coated with DNA (Birch 1997;Hansen & Wright 1999).
However, we suggest that the cheapest and simplest method is using A. tumefaciens.
However, those tissue culture based-methods are suggested to be time consuming and lead to soma clonal variation that affect both qualitative and quantitative characters of the plants (Labra et al. 2004). The direct transformation method without any tissue culture steps is termed as in-planta transformation (Feldmann & Marks 1987).
The production of a large number of uniform plants in a short time, less labor efforts, and minimal reagents requirements are some of main the advantages of the in-planta transformation system (Bent 2000). In addition, tissue culture-based transformation methods require carefulness to maintain sterile condition, a common difficulty in the tissue culture work.
We explored in-planta transformation of a table grape in order to provide a simple method of transformation that may be easier to be done compared to the in-vitro method.
In-planta transformation method has been done for Arabidopsis by applying Agrobacterium to the Arabidopsis seeds as the target of transformation (Feldmann 1992;Feldmann & Marks 1987) and a method of "clip 'n squirt" using inflorescences as targeted cells (Chang et al. 1994;Katavic et al. 1994). In-planta transformation method mediated with Agrobacterium was also reported for Brassica napus L. (Li et al. 2010), wheat (Razzaq et al. 2011), pommelo (Citrus maxima) (Zhanga et al. 2017) and Phalaenopsis orchid (Semiarti et.al. 2014), but there is a lack of report for table grape.
The current research explored two methods using A. tumefaciens with non-leafy shoots (shoots whose leaves were removed) as targeted cells, namely 'dipping method' and 'sweeping method'. The aim of the research was to investigate a simple method of inplanta transformation for the table grape.
The current method would be easier to be done and may be adopted for other species.

MATERIALS AND METHODS
We used a Sucrose Phospate Syntase (S0SPS1) gene that was obtained from Prof. The non-leafy shoots were then used as the target of the in-planta transformation (Fig. 2).
We explored two methods in the current research, namely the "dipping method" and "sweeping method". In the dipping method, the non-leafy shoots were dipped with the Agrobacterial suspension for 120 seconds (Fig. 3).
However, for the "sweeping method", the scars were swept with the Agrobacterial suspension using a cotton bud   (2) months after the treatment (Fig. 5).   To ensure the results, then we did PCR for the other putative transformed plants. Two plants were from the sweeping method and one was from the dipping method. The PCR result is shown in Fig. 8.
The second PCR analysis again confirmed that the transgene was inserted into the plant genome of the sweeping method plant, but it did not occur with those of the dipping method. The result is in the Table 1. The data in the Table 1 suggested that the S0SPS1 gene might be inserted in to the plant genome and was functionally worked on the putative vines. As we know that the Sucrose Phosphate Synthase is a key enzyme for sucrose biosynthesis in plants (Bruneau et al., 1991). However, we need further research to increase immersion time to allow the chance of gene integration. We also need to confirm the cimeras and ensure that the gene was integrated in the whole plant.
Transferred DNA (T-DNA) enters the plant as a single stranded molecule (Stachel et al. 1986;Tinland et al. 1994;Yusibov et al. 1994) that may eventually integrate into the nuclear genome. Although T-DNA integration is random (Kim et al. 2007), the mechanism of integration remains unclear (Park et al. 2015). However, irradiated protoplasts show a higher DNA integration frequency than do non-irradiated protoplasts (Kohler et al. 1989), suggesting that double stranded (ds)DNA damage sites could be targets of T-DNA integration (Park et al. 2015). Indeed, T-DNA molecules preferentially integrate into dsDNA break sites (Chilton and Que, 2003;Salomon and Puchta, 1998;Tzfira et al. 2003  Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol. Gen. Genet. 208: 1-9 Fujita, K., Matsuoka, T., Suzuki, S., and Takayanagi, T. (2009). In Planta Transformation Technique for