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Efficient Genetic Transformation of Vitis vinifera L. Mencía using a Hypervirulent Strain of Agrobacterium tumefaciens and qPCR Determination of Transgene Copy Number

Óscar Martínez, Elena Palomo-Ríos, Manuel Rey, María Victoria González
Am J Enol Vitic.  2024  75: 0750020  ; DOI: 10.5344/ajev.2024.24012

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Figures

  • Kanamycin selection strategies used in this study. Transformed somatic embryo aggregates were cultured in selection media composed of induction medium supplemented with 250 mg/L timentin and the indicated kanamycin concentrations (mg/L). The selection strategies were a standard strategy (strategy 1), where aggregates were cultured in selection medium with 50 mg/L kanamycin for 5 wk; an incremental strategy (strategy 2), with 25 mg/L kanamycin for 4 wk followed by 50 mg/L kanamycin for 1 wk; a progressively increasing kanamycin concentration strategy (strategy 3), with 6, 12, 25, 50, and 50 mg/L kanamycin for 1 wk each; and no initial selection strategy (strategy 4), with no kanamycin for 2 wk followed by 50 mg/L kanamycin for 3 wk. After 5 wk, treated somatic embryo aggregates were maintained in the presence of 50 mg/L kanamycin in all strategies.
    Figure 1

    Kanamycin selection strategies used in this study. Transformed somatic embryo aggregates were cultured in selection media composed of induction medium supplemented with 250 mg/L timentin and the indicated kanamycin concentrations (mg/L). The selection strategies were a standard strategy (strategy 1), where aggregates were cultured in selection medium with 50 mg/L kanamycin for 5 wk; an incremental strategy (strategy 2), with 25 mg/L kanamycin for 4 wk followed by 50 mg/L kanamycin for 1 wk; a progressively increasing kanamycin concentration strategy (strategy 3), with 6, 12, 25, 50, and 50 mg/L kanamycin for 1 wk each; and no initial selection strategy (strategy 4), with no kanamycin for 2 wk followed by 50 mg/L kanamycin for 3 wk. After 5 wk, treated somatic embryo aggregates were maintained in the presence of 50 mg/L kanamycin in all strategies.

  • Effect of kanamycin concentration on growth of Mencía grapevine somatic embryo aggregates in induction medium. A) Variation in fresh weight (final weight − initial weight; mean ± standard error) of somatic embryo aggregates after 4 (white) and 8 (black) wk of culture in induction medium with different concentrations of kanamycin. B) Growth of somatic embryo aggregates in induction medium for 8 wk after treatment with different concentrations of kanamycin. In (A), different letters within the same time of culture (uppercase for data collected after 4 wk of culture, lowercase for data after 8 wk) indicate statistically significant differences between kanamycin concentrations (p < 0.05).
    Figure 2

    Effect of kanamycin concentration on growth of Mencía grapevine somatic embryo aggregates in induction medium. A) Variation in fresh weight (final weight − initial weight; mean ± standard error) of somatic embryo aggregates after 4 (white) and 8 (black) wk of culture in induction medium with different concentrations of kanamycin. B) Growth of somatic embryo aggregates in induction medium for 8 wk after treatment with different concentrations of kanamycin. In (A), different letters within the same time of culture (uppercase for data collected after 4 wk of culture, lowercase for data after 8 wk) indicate statistically significant differences between kanamycin concentrations (p < 0.05).

  • Selection and recovery of transformed Mencía grapevine embryogenic material from somatic embryo aggregates cultured in induction medium supplemented with 50 mg/L kanamycin (selection strategy 1, Figure 1). A) Formation of potentially transformed grapevine Mencía somatic embryos after 2 mo of culture in the presence of kanamycin. B) Growth of Mencía grapevine somatic embryo aggregates after 3 mo of culture in the presence of kanamycin. Bars: 1 mm (A); 1 cm (B).
    Figure 3

    Selection and recovery of transformed Mencía grapevine embryogenic material from somatic embryo aggregates cultured in induction medium supplemented with 50 mg/L kanamycin (selection strategy 1, Figure 1). A) Formation of potentially transformed grapevine Mencía somatic embryos after 2 mo of culture in the presence of kanamycin. B) Growth of Mencía grapevine somatic embryo aggregates after 3 mo of culture in the presence of kanamycin. Bars: 1 mm (A); 1 cm (B).

  • Effect of selection strategy on the transformation efficiency of Mencía grapevine somatic embryo aggregates, indicated as the percentage of embryos showing response after selection treatment. The selection strategies tested were a standard strategy (strategy 1); an incremental strategy (strategy 2); a progressively increasing kanamycin concentration strategy (strategy 3); and no initial selection strategy (strategy 4). After 5 wk, treated somatic embryo aggregates were maintained in the presence of 50 mg/L kanamycin in all strategies. (A) Percentage of aggregates with embryogenic response (mean ± standard error) after each selection strategy. Different letters indicate statistically significant differences (p < 0.05). (B) Percentage of positive (black; GUS+) or negative (grey; GUS−) for the β-glucuronidase (GUS) histochemical analysis of the transformed embryogenic lines generated in each selection strategy.
    Figure 4

    Effect of selection strategy on the transformation efficiency of Mencía grapevine somatic embryo aggregates, indicated as the percentage of embryos showing response after selection treatment. The selection strategies tested were a standard strategy (strategy 1); an incremental strategy (strategy 2); a progressively increasing kanamycin concentration strategy (strategy 3); and no initial selection strategy (strategy 4). After 5 wk, treated somatic embryo aggregates were maintained in the presence of 50 mg/L kanamycin in all strategies. (A) Percentage of aggregates with embryogenic response (mean ± standard error) after each selection strategy. Different letters indicate statistically significant differences (p < 0.05). (B) Percentage of positive (black; GUS+) or negative (grey; GUS−) for the β-glucuronidase (GUS) histochemical analysis of the transformed embryogenic lines generated in each selection strategy.

  • β-glucuronidase (GUS) histochemical assay in Mencía grapevine somatic embryo transformed lines and regenerated plantlets. (A) Asynchronic somatic embryo aggregates cultured in DM1 differentiation medium. (B) Leaves of plantlets not transformed (left) and transformed (right) grown in conversion medium. Bars: 1 mm (A); 1 cm (B).
    Figure 5

    β-glucuronidase (GUS) histochemical assay in Mencía grapevine somatic embryo transformed lines and regenerated plantlets. (A) Asynchronic somatic embryo aggregates cultured in DM1 differentiation medium. (B) Leaves of plantlets not transformed (left) and transformed (right) grown in conversion medium. Bars: 1 mm (A); 1 cm (B).

  • Relative expression of the uidA gene in Mencía grapevine somatic embryo transformed lines. qPCR data were determined and statistically analyzed (p < 0.05) using REST-2009 with PCR efficiency correction and normalization with two reference genes, then compared with the 2−ΔΔCq method. GAPDH(m) and EF1-α(m) were used as reference genes for normalization and the transformed line with lowest transcript level (T23) was used as the calibrator. The average values of two independent experiments are represented ± the standard error.
    Figure 6

    Relative expression of the uidA gene in Mencía grapevine somatic embryo transformed lines. qPCR data were determined and statistically analyzed (p < 0.05) using REST-2009 with PCR efficiency correction and normalization with two reference genes, then compared with the 2−ΔΔCq method. GAPDH(m) and EF1-α(m) were used as reference genes for normalization and the transformed line with lowest transcript level (T23) was used as the calibrator. The average values of two independent experiments are represented ± the standard error.

Tables

  • Table 1

    Primer sources and sequences, amplicon lengths, and efficiency for qPCR assays. Efficiency was determined with LinRegPCR software and the indicated value represents the average of all qPCRs performed.

    Table 1
  • Table 2

    Transformation efficiency after additional selection in liquid medium. Transformed Mencía somatic embryo aggregates treated with each selection strategy in solid media were cultured in liquid induction medium supplemented with 50 mg/L kanamycin for 5 wk. The final transformation efficiency was calculated as the percentage of embryogenic response observed in each of the initial selection strategies, multiplied by the percentage embryogenic response after selection in liquid medium.

    Table 2
  • Table 3

    Estimation of the number of copies of transgenes nptII and uidA based on the values (mean ± standard error) of ΔCq and modified ΔCq in lines of transformed grapevine somatic embryo aggregates. The grapevine single-copy genes NCED2 and chi were used as the endogenous control genes.

    Table 3
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Efficient Genetic Transformation of Vitis vinifera L. Mencía using a Hypervirulent Strain of Agrobacterium tumefaciens and qPCR Determination of Transgene Copy Number
Óscar Martínez, Elena Palomo-Ríos, Manuel Rey, María Victoria González
Am J Enol Vitic.  2024  75: 0750020  ; DOI: 10.5344/ajev.2024.24012
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