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

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.

Agrobacterium-mediated genetic transformation of Chinese chestnut (Castanea mollissima Blume)

“…Growers eagerly expect for excellent cultivars possessing improved traits. Genetic transformation is a powerful tool for the genetic improvement of plants, and it has been successfully applied to many woody plants, such as European chestnut, American chestnut, apple and grape (Seabra and Pais 1998; Yepes et al 1994;Andrade et al 2009;Sabbadini et al 2019). In this study, precise genetic transformation of Chinese chestnut was established, which will benefit its application in validating gene function and molecular breeding of Chinese chestnut.…”

“…Kanamycin is one of the most commonly used antibiotics to kill untransformed tissue in many genetic transformation processes (Mcgranahan et al 1993;Seabra and Pais 1998;Yepes and Aldwinckle 1994;Sabbadini et al 2019). The concentration of kanamycin differs among species, such as 100 mg/L for American pecans (Mcgranahan et al 1993), 70 mg/L for grape (Sabbadini et al 2019) and 150 mg/L for European chestnut (Seabra and Pais 1998). The 150 mg/L kanamycin used in the genetic transformation of European chestnut suppressed the regeneration of 98% of non-transformed somatic embryos (Seabra and Pais 1998).…”

“…4k, l), which were considered to be chimeras. The low transgenic regeneration rate of Chinese chestnut embryogenic calli presumably was because of addition of kanamycin, which reduced shoot regeneration capacity (Sabbadini et al 2019). In addition to antibiotics, the Agrobacterium strain may affect the emergence of shoots.…”

Biosafety of GM Crop Plants Expressing dsRNA: Data Requirements and EU Regulatory Considerations

“…Several studies demonstrated the transfer of siRNAs from the transgenic rootstock to the nontransgenic scion conferring resistance to virus in fruit trees (Zhao and Song, 2014) and grafted cucumber (Bai et al, 2016). This technology is now studied also for conferring resistance to pests (Taning et al, 2020) and diseases (Sabbadini et al, 2019). Such systemic silencing probably depends on amplification of siRNAs through secondary siRNA production.…”

Biotechnological Approaches: Gene Overexpression, Gene Silencing, and Genome Editing to Control Fungal and Oomycete Diseases in Grapevine

“…However, the application of the HIGS approach is limited by poor public acceptance and strict legislative rules applied to GMO cultivation, and also by the lack of efficient in vitro regeneration and genetic transformation protocols for several crops, and more often, for all the genotypes within the same species [ 115 ].…”