Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis

S Sabbadini,A Ricci,L Capriotti,T Pandolfini,B Mezzetti,O Navacchi,B Molesini,C Limera

doi:10.1038/s41598-018-37335-7

S Sabbadini, A Ricci + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-37335-7

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Abstract

The success of in vitro plant regeneration and the competence of genetic transformation greatly depends on the genotype of the species of interest. In previous work, we developed a method for the efficient Agrobacterium-mediated genetic transformation via organogenesis of V. vinifera cultivar Thompson Seedless, by using meristematic bulk (MB) as starting tissue. In this study, we applied this method for the regeneration and transformation of MBs obtained from the Italian cultivar Ciliegiolo and two of the commonly used Vitis rootstocks, 110 Richter and Kober 5BB, in comparison with Thompson Seedless. The A. tumefaciens strain EHA105, harbouring pK7WG2 binary vector, was used for the transformation trials, which allowed selection through the enhanced-green fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII) gene. Putative transformed tissues and/or shoots were identified by either a screening based on the eGFP expression alone or its use in combination with kanamycin in the medium. MBs obtained from Thompson Seedless showed the highest regeneration and transformation cell competence, which subsequently allowed the recovery of stably transformed plants. Ciliegiolo, 110 Richter, and Kober 5BB, produced actively growing transgenic calli showing eGFP fluorescence, more consistently on selective media, but had no regenerative competence.

Highlights

Genetic engineering of grapevine represents a powerful alternative to conventional plant breeding programs, offering the possibility of introducing useful agronomic traits in cultivars and/or rootstocks without altering their desirable characteristics
It was not possible to obtain similar consistently proliferating transformed calli from meristematic bulk (MB) tissues cultured on non-selective media, probably due to the absence of selection pressure normally conferred by kanamycin on the non-transformed cells
It seems that the transformation efficiency is not linked to the initial regeneration potential; MB slices of Ciliegolo and Kober 5BB didn’t produce any transgenic shoots after transformation, they showed regeneration efficiency comparable to that of Thompson Seedless before Agrobacterium infection

Summary

Introduction

Genetic engineering of grapevine represents a powerful alternative to conventional plant breeding programs, offering the possibility of introducing useful agronomic traits in cultivars and/or rootstocks without altering their desirable characteristics. The use of anther filaments as a starting material for somatic embryogenic calli induction requires high expertise for the selection of the correct flower stage and is characterized by several drawbacks, such as time consuming and laborious processes for the creation and maintenance of the embryogenic cultures This method is not applicable to some grapevine genotypes[5]. The use of non-destructive reporter genes alone or combined with antibiotic-based selection system might ease the identification of escapes and chimera, which are commonly protected when the selection system relies solely on the presence of antibiotics in the culture medium This phenomenon has often been associated with the persistence of antibiotic-resistant A. tumefaciens cells, and/or to the presence of transformed plant cells close to non-transformed ones with a supposed detoxifying effect in the surrounding area[24,29,30,31,32]. We compared the combined use of the nptII gene and eGFP visual screening to the use of eGFP as a unique means for the recovery of transformed events and plants obtained via organogenesis

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Results

Conclusion