An Improved Agrobacterium-Mediated Transformation Method for an Important Fresh Fruit: Kiwifruit (Actinidia deliciosa)

Abstract

Genetic transformation is an essential tool for investigating gene function and editing genomes. Kiwifruit, recognized as a significant global fresh fruit crop, holds considerable economic and nutritional importance. However, current genetic transformation techniques for kiwifruit are impeded by low efficiency, lengthy culture durations (a minimum of six months), and substantial labor requirements. In this research, we established an efficient system for shoot regeneration and the stable genetic transformation of the 'Hayward' cultivar, utilizing leaf explants in conjunction with two strains of Agrobacterium that harbor the expression vector pBI121-35S::GFP, which contains the green fluorescent protein (GFP) gene as a visible marker within the T-DNA region. Our results show that 93.3% of leaf explants responded positively to the regeneration medium, producing multiple independent adventitious shoots around the explants within a six-week period. Furthermore, over 71% of kanamycin-resistant plantlets exhibited robust GFP expression, and the entire transformation process was completed within four months of culture. Southern blot analysis confirmed the stable integration of GFP into the genome, while RT-PCR and fluorescence microscopy validated the sustained expression of GFP in mature plants. This efficient protocol for regeneration and transformation provides a solid foundation for micropropagation and the enhancement of desirable traits in kiwifruit through overexpression and gene silencing techniques.

Keywords: Southern blot analysis; high-efficiency genetic transformation; kiwifruit (Actinidia deliciosa); leaf explant; mature plant; stable GFP expression.

Figure 1

Plant regeneration from leaf explants of kiwifruit. (A) Shoot induction from leaf explants on 3/4 MS+ 5 mg/L BA + 1 mg/L Zt + 0.15 mg/L IBA medium (SIM) after 4 weeks of culture. (B) Root induction from regenerated shoots on 1/2 MS+ 0.25 mg/L IBA medium (RIM) after 6 weeks of culture.

Figure 2

Observation of GFP expression at different steps in Agrobacterium-mediated transformation of kiwifruit. (A) Kanamycin-resistant small swellings emerged on wounded leaf segments on solid SM after 10 days of culture. (B) Protuberance formation on kanamycin-resistant swellings on solid SM after 3 weeks of culture. (C) Kanamycin-resistant small shoots emerged on solid SM after 5 weeks of culture. (D) Normally grown kanamycin-resistant shoots on solid SM after 8 weeks of culture. (E) GFP observation in kanamycin-resistant small swellings (A). (F) GFP observation in kanamycin-resistant protuberances (B). (G) GFP observation in kanamycin-resistant small shoots (C). (H) GFP observation in kanamycin-resistant plantlets (D). Bar = 1 mm.

Figure 3

PCR analysis of transformed kiwifruits. Genomic DNA was extracted from fresh leaves of pot-grown wild-type (WT) plants and ten putative GFP-positive transgenic kiwifruit lines. (A) Rooted transgenic kiwifruit plant exhibiting GFP fluorescence after 6 weeks of culture on RIM supplemented with 50 mg/L kanamycin. (B) PCR detection of GFP transgene in ten independent transgenic lines using gene-specific primers (Table S3). WT: Non-transformed wild-type kiwifruit. GFP1–5: Five independent GFP-positive kiwifruits transformed with EHA105/pBI-35S::GFP. GFP6–10: Five independent GFP-positive kiwifruits transformed with GV3101/pBI-35S::GFP. M: DL1000 DNA marker (Takara, Japan). R: root; L1: First true leaf; AM: Apical meristem.

Figure 4

A Southern blot analysis of GFP integration in transformed kiwifruits. DNA from a non-transformed plant and seven GFP-positive transgenic kiwifruits was digested with BamHI, fractionated using electrophoresis, transferred to a nylon membrane, and hybridized with the full-length GFP probe. WT: a non-transformed wild-type kiwifruit. GFP1–4: four independent transgenic kiwifruits transformed with EHA105/pBI-35S::GFP. GFP6–8: three independent transgenic kiwifruits transformed with GV3101/pBI-35S::GFP. M: DL 10,000 DNA marker (Takara, Japan). P: plasmid pBI-35S::GFP.

Figure 5

GFP observation from WT and three transgenic kiwifruits grown in greenhouse. (A) Three-month-old WT and three transgenic kiwifruits grown in greenhouse. (B) Detection of GFP expression from WT and three transgenic kiwifruits (A) by RT–PCR. (C) Observation of GFP expression from mature leaves of WT and three transgenic kiwifruits (A) using fluorescence microscopy. Bar = 1 mm.

Figure 6

Summary of procedure for Agrobacterium-mediated genetic transformation of kiwifruit. Timetable is displayed on panel.