The Supervirulence Plasmid pToK47 from Agrobacterium tumefaciens A281 Improves Transformation Efficiency of Hevea brasiliensis

Problem statement: The present study investigates the ability of the p Tok47 supervirulence plasmid form Agrobacterium tumefaciens A281 to enhance genetic transformation in Hevea brasiliensis. Approach: Hevea anther callus was transformed via Agrobacterium-mediated genetic transformation using two strains of Agrobacterium (GV2260 and GV3850) harboring the human serum albu min cDNA and the supervirulent plasmid pToK47 from Agrobacterium tumefaciens A281. The transformed callus was selected using kanamycin as the election agent Results: The Agrobacterium strain GV2260 benefited from the presence of the su pervirulence plasmid in giving a higher frequency of 7.4% transformed callus, 344.8% embryogenesis an d 11.6% plantlet production compared to the corresponding strain on its own giving 0.9% transfo rmed callus, 204.5% embryogenesis and 4.4% plantlet production. Similarly, Agrobacterium strain GV3850 conferred a higher frequency using th e supervirulent plasmid, resulting in 3.5% transforme d callus, 138.5% embryogenesis and 3.5% plantlet production compared to the corresponding strain on its own giving 0.7% transformed callus, 137.5% embryogenesis and 9.0% plantlet production. These f indings were confirmed by the Wilcoxon Signed Rank Test that compared the effectiveness of the su p rvirulence plasmid in increasing the rate of genetic transformation in the calli surviving in ka namycin growth medium for GV2260 (p<0.001) and for GV3850 (p<0.05). Conclusion: This study showed that both Agrobacterium strains benefited from the presence of the supervirulence plasmid in givin a higher frequency of transformed callus, embryoids and plantlets. These results suggest that a ighly virulent binary vector system might prove especially useful in generating high frequency tran sformation of Hevea.


INTRODUCTION
The main challenge with genetic transformation of tree species is achieving high transformation efficiency for desired clones or cultivars. In this respect, it is important to have an efficient tissue culture system to obtain plantlets from cells containing the transgene. In the case of Hevea brasiliensis (rubber tree), the highly embryogenic clone GL1 is routinely used as a vehicle for genetic transformation.
Agrobacterium tumefaciens harbors a tumorinducing plasmid (Ti plasmid) and the region of T-DNA within this plasmid encodes for the expression of the plant growth regulator genes for auxin and cytokinin, which leads to the formation of tumors in plant cells [1,2] . The Ti plasmid also harbors another important segment, the virulence (vir) region and this encodes most of the functions necessary for T-DNA transfer to plant cells [3] .
In A. tumefaciens A281 (the carrier of the plasmid pTiBo542) the tumors formed after infection with plant cells were larger, were early-appearing and the tumurogenesis applied to a wide range of plants, compared to other A. tumefaciens strains [4] . For the above reason, the plasmid pToK47 was constructed by subcloning a 15.8 kb fragment of pTiBo542 that carries the entire virB, virG and virC operons. When mobilized into Agrobacterium strains A348, A281, A208 and T37 all the resulting strains displayed larger tumor formation compared with corresponding wild type strains [4] .
Several publications have shown that transformation of previously recalcitrant species of monocotyledons (rice, corn and maize) can be accomplished through the use of disarmed A. tumefaciens strains to which additional copies of certain virulence genes were added [5,6] and also showed an improved procedure for production of white spruce using Agrobacterium containing the virulence regions from pToK47 [7,8] .
In the present investigation, an efficient transformation protocol for Hevea was developed using the supervirulence plasmid pToK47 containing virB, virC and virG genes from Agrobacterium tumefaciens A281.

Agrobacterium strains and plasmid vectors:
The binary vector pLGMR.HSA was constructed by inserting the expression cassette containing the 35SCaMV promoter, the multicloning sites and the CaMV Poly A tail from pJIT62 (Guerineau, France unpublished) into the polylinker of pBIN19 [9] at KpnI and EcoRV sites. The gene segment coding for Human Serum Albumin (HSA) was amplified by Polymerase Chain Reaction (PCR). The PCR product (1.8 kb) was cloned into the SmaI site of pLGMR at the multicloning sites to yield the expression vector pLGMR. HSA [10] .

Agrobacterium-mediated transformation:
Hevea anther callus (clone GL1) was transformed using GV2260(pLGMR.HSA:pToK47) or GV3850(pLGMR.HSA:pToK47) as described [12] . For transformation experiments, a total of 2332 anther callus derived from individual anthers of GL1 cultivar were immersed for 1 min in Agrobacterium suspension for the two strains with and without pToK47. A total of 16 individual experiments were performed with an equal number of anther callus ranging from 75-210. After immersion of the calli in an Agrobacterium suspension for 1 min, the calli were blotted dry using sterile Whatman No 1 filter paper and then transferred to initiation medium [13] for a co-cultivation period of 2 days. The calli were then transferred to fresh initiation medium containing cefotaxime (Duchefa, Holland) at 300 µg mL −1 and ticarcillin (Duchefa, Holland) at 300 µg mL −1 to eliminate Agrobacterium infection.
Selection of transformants and regeneration into plantlets: After 1 week without selection, calli were transferred to fresh initiation medium containing 50 µg mL −1 kanamycin. The calli were transferred to fresh initiation medium every 2 weeks and the selection on kanamycin was increased from 50-100 µg mL −1 in the third selection. The concentrations of cefotaxime and ticarcillin were reduced from 300-150 µg mL −1 and the finally to a concentration of 100 µg mL −1 in the third selection. Calli that were kanamycin resistant was isolated and subcultured onto fresh initiation medium for 45-50 d, after which the calli were transferred to differentiation medium [13] containing 100 µg mL −1 kanamycin for a period of 2 months. Kanamycinresistant embryoids were generated and transferred to developmental medium [13] for plantlet production.

Statistical analysis:
In data analysis, variation due to day to day effects were removed by pairing treatments with and without pToK47. As Gaussian distribution of the data was suspect, a nonparametric paired test, the Wilcoxon Matched Pairs Signed Rank Test, was used in statistical evaluation of the effectiveness of the supervirulence plasmid on transformed (i.e., kanamycin-resistant) calli and transformed embryos.

DNA analysis in putative transformants of Hevea:
PCR analysis was used to detect the presence of NPTII and HSA in the kanamycin-resistant callus tissues. Genomic DNA from independently transformed and untransformed (control) tissues was subjected to PCR. Fig. 1 shows that the samples from transformed tissues gave the predicted DNA fragment bands of 0.7 kb for NPTII gene (lanes 3-8), Fig. 1a and 1.8 kb for HSA  gene (lanes 3-8), Fig. 1b, whereas no amplification was detected in the sample from untransformed tissue.  Southern analysis with the DIG HIGH-PRIME labeled NPTII and HSA probe showed a hybridization signal in the six transformed tissues analyzed (lanes 3-8) (Fig. 2a and b). These results show that sustained growth and development of the callus culture on kanamycin medium is a good indicator of successful genetic transformation.
Effectiveness of the supervirulence plasmid (pToK47) in GV2260 and GV3850 Agrobacterium strains: The most appropriate indicator of the effectiveness of the supervirulence plasmid in increasing the rate of genetic transformation is in the calli surviving in kanamycin growth medium. This is a direct measure of the rate of transfomation success. This finding was confirmed by the Wilcoxon Signed Rank Test that compared the results from each culture initiation day (p<0.001 for GV2260 (Table 1) p<0.05 for GV3850 (Table 2)).  While scorings of transformed embryoids or plantlets that develop subsequently might also reflect, to some extent, the role of the supervirulence plasmid, these indicators are not as accurate. Individual callus, once transformed with or without the help of the supervirulence plasmid are subsequently multiplied and the final number of transformed calli might not be exactly proportional to the number of original transformants. Moreover, some of transformed calli would be more successful than others in giving rise to multiple embryoids and plantlets later. Notwithstanding this, the numbers of embryoids and plantlets obtained from callus cultures with the supervirulence plasmid was higher in both GV2260 (Table 1) and GV3850  (Table 2), this advantage is statistically significant in GV2260 (Table 1).