*3.1. Effect of Gamma Radiation on In Vitro Survival and Development*

The different doses of gamma irradiation showed a significant effect on survival percentage, number of shoots per explant, and length and number of leaves per shoot (Figure 1). The LD50 was observed at the 60 Gy, this dose reduced survival of explants by 52%. The highest survival percentage was observed at the 0 and 20 Gy doses, with 100% survival, whereas the lowest survival percentages were obtained at the 80 and 100 Gy doses, with 31 and 24% survival, respectively (Figure 1a). Regarding the number of shoots per explant, the highest number of new shoots was obtained at the 20 and 40 Gy doses, with 6 and 5.3 shoots per explant, respectively, whereas the lowest number of shoots was observed at the 60, 80 and 100 Gy doses, obtaining only 3 shoots per explant (Figure 1b). The longest shoot length was obtained at the 0 and 20 Gy doses, with 3.1 and 3 cm in length, respectively. The shortest length was observed at the 60, 80 and 100 Gy doses, with 1.6 to 1.8 cm in height (Figure 1c). The highest number of leaves was observed at the 0 and 20 Gy doses, with 3.3 and 3 leaves per shoot, whereas the lowest number of leaves per shoot was observed at 60, 80 and 100 Gy, with 1.6 to 1.7 leaves per shoot on average (Figure 1d). In addition, the administration of different doses of gamma irradiation had an effect on the multiplication rate in vanilla shoot development (Figure 2).

**Figure 1.** Effect of gamma radiation on in vitro survival and development of vanilla (*Vanilla planifolia* Jacks. ex Andrews). (**a**) Survival percentage, (**b**) shoots per explant, (**c**) shoot length and (**d**) leaves per shoot at 60 d of culture. Values represent the mean ± standard error. Means with different letters are significantly different (Tukey, *p* < 0.05).

**Figure 2.** Effect of different doses of gamma irradiation on in vitro shoot development of vanilla (*Vanilla planifolia* Jacks. ex Andrews) at 60 days of culture; (**a**–**f**) 0, 20, 40, 60, 80 and 100 Gy, respectively. Black bar = 1 cm.

#### *3.2. DNA Polymorphism of Gamma Radiation on Somaclonal Variation*

The ISSR analysis revealed the presence of monomorphic and polymorphic bands between the control and the different irradiation doses with respect to the donor plant (Figure 3 and Supplementary Figure S1). The eight selected ISSR primers amplified a total of 43 bands ranging from 250 to 2000 bp. The primers that showed the highest percentage of polymorphism were UBC-808 with 42.6% (9 bands), followed by UBC-836 with 34.7% (6 bands) and UBC-840 with 28.7% (6 bands). On the other hand, the primers that showed less than 15% polymorphism were UBC-825 (6 bands), T06 (4 bands) and UBC-809 (7 bands). Primers T05 (1 band) and UBC-812 (4 bands) revealed the presence of monomorphic fragments (Table 1).

**Figure 3.** Electrophoresis pattern with ISSR markers of ten individuals (1–10) of vanilla (*Vanilla planifolia* Jacks. ex Andrews) exposed to gamma radiation with respect to donor plant (D). (**a**) Dose 60 Gy, primer UBC-812 (**b**) dose 20 Gy, primer UBC-840. M = molecular weight marker and bp = base pairs.

The dendrogram based on the neighbor joining model showed no similarity in the irradiation doses evaluated with respect to the donor plant. According to Bootstrap probabilities, similarity distances and branch lengths, the first group was considered to be the donor plant, whereas the second group comprised the doses of 0, 20, 40, 60, 80 and 100 Gy. The second group was divided into six subgroups, where subgroup six had the greatest similarity distance (0.76), formed by nine individuals of 100 Gy doses. The shortest distance was observed in the first and second subgroups with a distance of 0.88 and 0.86, respectively, with one individual of 0 and 20 Gy, respectively. For the rest of the subgroups, no clustering trend was observed with respect to the gamma radiation doses evaluated (Figure 4).

**Figure 4.** Neighbor joining dendrogram based on Jaccard's coefficient, calculated for each gamma irradiation dose with respect to the donor plant of vanilla (*Vanilla planifolia* Jacks. ex Andrews). The values under line represent: 1 similarity, and 0 divergence. I-VI: subgroups with different similarity distances.
