Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward

Abstract

Almost 30 years have passed since the first publication reporting regeneration of transformed peach plants. Nevertheless, the general applicability of genetic transformation of this species has not yet been established. Many strategies have been tested in order to obtain an efficient peach transformation system. Despite the amount of time and the efforts invested, the lack of success has significantly limited the utility of peach as a model genetic system for trees, despite its relatively short generation time; small, high-quality genome; and well-studied genetic resources. Additionally, the absence of efficient genetic transformation protocols precludes the application of many biotechnological tools in peach breeding programs. In this review, we provide an overview of research on regeneration and genetic transformation in this species and summarize novel strategies and procedures aimed at producing transgenic peaches. Promising future approaches to develop a robust peach transformation system are discussed, focusing on the main bottlenecks to success including the low efficiency of A. tumefaciens-mediated transformation, the low level of correspondence between cells competent for transformation and those that have regenerative competence, and the high rate of chimerism in the few shoots that are produced following transformation.

Keywords: Rosaceae; biotechnology; organogenesis; plant breeding; somatic embryogenesis; stone fruits.

Figure 1

Direct adventitious regeneration from immature peach cotyledons: (a–c) Bud regeneration observed in immature cotyledons of “Starlite”, “Bailey”, and “Guardian”, respectively, under no selection regime and controls after 5–6 weeks from the beginning of the experiment (bar = 0.5 cm). (d) Rooted shoots after 4 weeks in rooting medium prepared for acclimatization (bar = 1 cm). (e) Potted plant cultured in a greenhouse after the rooting and acclimatization process. (f) Scheme of the methodology followed for regeneration of transformed shoots.

Figure 2

Factors affecting adventitious regeneration from peach mature seed hypocotyl slices. (a) First buds appearing after 4 weeks of culture from the beginning of the experiment (bar = 1 mm). (b) Adventitious regeneration from a peach hypocotyl section after 6 weeks of culture from the beginning of the experiment (bar = 1 mm). (c) Effect of silver thiosulphate (STS) on regeneration. A total of 182, 498, 862, and 700 explants were used in this experiment for “Bounty”, “Lovell”, “Nemaguard”, and “Bell of Georgia”, respectively. Vertical bars indicate standard errors (SE). (d) Effect of 2-aminoethoxyvinyl glycine (AVG) on regeneration. A total of 229 and 484 explants were used in this study for “Nemaguard” and “Bell of Georgia”, respectively. Vertical bars indicate SE. Asterisks indicate significant regeneration increased (p < 0.01) compared to the control without addition of AVG, according to Pearson’s chi-test. (e) Effect of dark incubation period on regeneration. A total of 255 and 326 explants were used in this study for “Nemaguard” and “Bell of Georgia”, respectively. Vertical bars indicate SE. Asterisks indicate statistical significance (p < 0.01) compared to the treatment without dark induction, according to Pearson’s chi-test. All the experiments were repeated at least twice.

Figure 3

Effect of aminoglycoside antibiotics (paromomycin and kanamycin) on mature peach seed hypocotyl sections. (a) Effect on adventitious bud regeneration. For this study, 450 explants were used (cv. “Bell of Georgia”). The experiment was repeated at least twice. Bars indicate SE. (b) Explants incubated in regeneration medium containing 20 mg/L of the specified antibiotic after 5 weeks of culture (bar = 0.5 cm).

Figure 4

Effect of BASTA herbicide on mature peach seed hypocotyl sections. Color column charts represent explant survival (%) after 2 weeks from the beginning of the experiment. Line charts represent regeneration rates (%) after 7 weeks from the beginning of the experiment with the vertical bars indicating SE. A total of 177, 264, 132, and 183 explants were used in this experiment for “Nemaguard”, “Bell of Georgia”, “Lovell”, and “Bounty”, respectively. Experiments were repeated at least twice.

Figure 5

Regeneration and transformation from peach seed-derived internode explants. (a) Histological study of the internode/cotyledon attachment area at day 0 (bar = 1 mm). (b) Histological study of the internode/cotyledon attachment area at day 9. Internode with axillary bud with evident meristematic dome with leaflets growing from the epidermis (arrow) (bar = 1 mm). (c) Adventitious shoot regeneration from a peach internode explant (bar = 1 mm). (d) Adventitious shoot regeneration from a EHA101 pVNFbin-infected explant cultured in regeneration medium supplemented with 20 mg/L paromomycin (bar = 1 mm). (e) β-Glucuronidase (GUS) activity in a peach internode explant (bar = 1 mm). (f) Green fluorescent protein (GFP) activity in a peach internode explant (bar = 1 mm).

Figure 6

Somatic embryogenesis (SE) trials on peach mature explants. (a) In vitro meristematic bulk (MB) of “Hansen 536”; the arrow indicates the type of young leaf collected and used as starting explant in SE induction experiment (bar = 1 cm). Brownish calli (arrow) developed from “Hansen 536” leaves cultured on medium C (b) and on medium D (c); the images were taken after 3 months from the beginning of the experiment (bar = 2 mm). Cream-colored calli (arrow) developed from “Hansen 536” leaves cultured on medium A (d), on medium B (e), and on medium E (f); the images were taken after 3 months from the beginning of the experiment (bar = 2 mm). Necrotic calli from “Hansen 536” leaves cultured on plant growth regulator (PGR)-free medium (g) after 4 months from the beginning of the experiment (bar = 1 cm). Cuttings of peach rootstock “GF677” (h) and sterile unopened flowers of “GF677” (i) used as starting explants in the SE induction experiment (bar = 1 cm). Cream-colored calli (arrow) developing from petal (j) and anther with filament (k) of “GF677”, both cultured on PAM medium after approximately 3 months from the beginning of the experiment (bar = 2 mm). (l) Cream-colored calli formation (arrow) from “GF677” anther with filament cultured on PIV medium [47] (bar = 2 mm). (m) “GF677” anthers with attached filament cultured on MSI medium [52] for 3 months (bar = 1 cm).

Figure 7

In vitro micropropagation of peach (P. persica) rootstock cv. “Bailey-OP”. (a) Shoot culture explant source from greenhouse-grown plant. Top (b) and bottom (c) views of an individual in vitro shoot cluster of peach rootstock “Bailey-OP” derived from a single shoot explant after 50 days of cultivation on LP medium supplemented with 4.5 µM 6-benzylaminopurine (BAP) and 0.5 µM IBA. Rooted shoot without (d,e) or with (f,g) exposure to VCs emitted by C. sphaerospermum isolate TC09 for 10 days. (h) Growth of plantlets previously treated without (tray on left, control) and with (tray on right) volatile compounds (VCs) 1 month after transplanting to soil in 1020 trays. In this representative comparison, control tray contains 36 surviving plants out of 100 transplanted plants. The tray on right side has 46 surviving plants out of 52 transplanted plants. (i) Normal growth and development of in vitro propagated “Bailey-OP” plants 3 months after transplanting.

Figure 8

Transient and stable GFP expression detected in peach (P. persica cv. “Bailey-OP”) shoot explants transformed with the binary vector pSGN. Detection of transient GFP expression in non-transformed (white light (a) and UV light (b)) and transformed (white light (c) and UV light (d)) shoot explants 1 week after transformation. Detection of stable GFP expression in callus tissue derived from control (white light (e) and UV light (f)) and transformed (white light (g) and UV light (h)) shoot explants after 1 month in selection with 100 mg/L kanamycin (bar = 2 mm).

Figure 9

Regeneration and transformation from peach nodal explants. (a) Effect of antibiotics on adventitious regeneration from non-infected explants and EHA101 (pVNFbin)-infected explants. Regeneration data were collected at 6 weeks from the beginning of the experiment. Asterisks indicate statistical significance (p < 0.01) compared to the “no selection” treatment according to Pearson’s chi-test. A total of 565 explant “Bailey-OP” were used for this study. Experiment was repeated at least twice. (b) Chimerical regenerated shoot showing GUS activity (arrow) (bar = 1 mm).

Figure 10

Organogenesis trials on peach meristematic bulks (MBs). (a) MB of “Hansen 536” (bar = 1 cm). (b) Slices (1 cm², 2 mm thick) obtained from “Hansen 536” MBs used as starting explants for A. tumefaciens-mediated transformation trials (bar = 1 cm). “Hansen 536” stably transformed callus-expressing eGFP (arrow) observed under UV light (c) or under white light (d). Photographs taken at 3 months post-infection (bar = 2 mm).