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Breeding objectives. Genetic transformation of Actinidia has focused on enhancing disease resistance, increased tolerance of drought and salinity, accumulation of bioactive compounds important in human health, improving rootstocks and manipulation of ethylene production in relation to fruit ripening (reviewed in Oliveira and Fraser, 2005; Table 1.1.2).

Table 1.1.2. Summary of achievements of Actinidia genetic transformation. Genotypes were corrected from the original papers and named according to the current taxonomy of the genus Actinidia (Ferguson, 2016).

PROTOCOL. Although most genetic transformations were Agrobacterium mediated, other methods, i.e. particle bombardment and PEG mediated, have been reported. Qiu et al. (2002) transformed A. deliciosa cell suspensions using particle bombardment. They introduced the maize DHN1 gene (induced in response to abiotic stresses) fused to the green fluorescent protein (GFP) reporter gene. GFP expression was localized in the nucleus after 10 h in response to increased osmotic stress.

PEG-mediated permeabilization yielded high levels of transient expression, especially in combination with heat shock (Oliveira et al., 1991). They used chloramphenicol acetyl transferase (CAT) reporter gene to assess the conditions for PEG-mediated transfection of kiwifruit protoplasts. The best CAT activity was obtained using 30% PEG with protoplasts that had been exposed to heat shock (45°C, 5 min); however, no transgenic plants were recovered. To recover transformants, selection pressure was applied only after recovery of colonies, and removed again to facilitate shoot multiplication and elongation (Oliveira and Raquel, 2001).

There are several critical points in the Agrobacterium-mediated transformation system that determines efficiency: genotype, explant type, Agrobacterium strain, cocultivation conditions and recovery/selection of the transformed cells. Although most of the transformation systems have been developed for A. deliciosa, A. chinensis and A. eriantha, there are also protocols for A. arguta, A. kolomikta and A. latifolia (reviewed in Wang and Gleave, 2012). Targeted explants are leaves (whole, discs or strips), petioles and, less frequently, stems.

Agrobacterium strains LBA4404, A281, C58, EHA101 and EHA105 have been used for transformation (Wang and Lin-Wang, 2007). Janssen and Gardner (1993) compared the efficiency of different Agrobacterium strains for transformation of A. deliciosa. In general, strain A281 produced slightly higher gene transfer rates than C58 and EHA101. Strain A281 harbours a tumour-inducing plasmid (pTiBo542) that contains an extra copy of a transcription activator involved in the regulation of the vir genes (Wang and Lin-Wang, 2007). Fraser et al. (1995) did not find marked differences between A281 and C58 strains (both carrying the pKIWI105 plasmid) in transformations of A. chinensis.

A comparison of four Agrobacterium strains (A281, GV3101, EHA105 and LBA4404) for transformation efficiency of A. chinensis was reported (Wang and Lin-Wang, 2007). The highest callus initiation from inoculated leaf strips was obtained with A281 strain (27%), followed by EHA105 (22.2%); however, <20% of the transformants from A281 developed shoots and roots. In contrast, >70% of calluses derived from strains EHA105, GV3101 and LBA4404 regenerated shoots and roots. With A. eriantha (Wang et al., 2006), similar results were obtained except that no shoot regeneration was observed from calluses induced after the transformation with strain A281.

Several reports (Wang et al., 2006; Han et al., 2010; Honda et al., 2011; Tian et al., 2011) demonstrated that time required for Agrobacterium infection and coculture were variable factors. The infection time for leaves ranged from 2 min (Tian et al., 2011) to 30 min (Wang et al., 2006; Han et al., 2010; Honda et al., 2011). The best coculture time was 2 days (Tian et al., 2011).

Janssen and Gardner (1993) and Matsuta et al. (1993) found that acetosyringone (AS) in the Agrobacterium growth and cocultivation medium increased β-glucuronidase expression in A. deliciosa leaves. Wang et al. (2006) improved the transformation efficiency of A. chinensis and A. eriantha by using AS in the bacterial culture medium before inoculation. However, Tian et al. (2011) showed a slight effect of AS on transformation efficiency.

Some species, e.g. A. arguta, show considerable browning and necrosis during shoot regeneration (Wang and Lin-Wang, 2007), and this may be caused by the additional stress during transformation and selection. Han et al. (2010) concluded that browning was significantly alleviated and plantlets could be regenerated using half-strength MS basal medium and low light intensity (3.4 μmol/m²/s). In general, the most common selection protocol employs 25–150 mg/l kanamycin, and Agrobacterium removal is usually achieved with timentin (300 mg/l), carbenicillin (200 mg/l) or cefotaxime (500 mg/l).

ACCOMPLISHMENTS. Koshita et al. (2008) transformed A. deliciosa with an anthocyanin regulatory gene of grapevine (VlmybA1-2) that can be used as reporter gene without any staining procedure. Enhanced salt tolerance by the overexpression of an Arabidopsis Na⁺/H⁺ antiporter gene (Tian et al., 2011) and the modification of plant architecture by the introduction of the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene (Honda et al., 2011) have been reported.

The chemical composition of the fruit has been altered to increase quality and to produce bioactive compounds. Kim et al. (2010b) increased the fruit content of β-carotene and lutein by introducing several carotenoid biosynthetic Citrus genes with a transformation protocol involving micro-cross sections of shoots (800 μm thick) as explants.

Functional genomic studies employing genetic transformation include both the elaboration of knockdown lines for the genes encoding a carotenoid cleavage dioxygenase (AcCCD8, Ledger et al., 2010) and a 1-aminocyclopropane 1-carboxylate (ACC) oxidase (AcACO1, Atkinson et al., 2011), as well as for the overexpression of the genes (SVP3 and SVP2) encoding SVP protein genes (Wu et al., 2014, 2017). Actinidia was also transformed to be used as a heterologous system for functional analysis of the DkMyb4 persimmon gene to study its function in the biosynthesis of condensed tannins (Akagi et al., 2009). The main goals and achievements of Actinidia transformation with genes of interest are summarized in Table 1.1.2.