**4. Discussion**

This study showed that gamma irradiation has an effect on survival and development during in vitro multiplication of *V. planifolia*. The mortality of the explants at doses higher than 60 Gy (DL50) could be explained by the radiosensitivity of explants exposed to gamma rays. The sensitivity of explants to gamma radiation depends on tissue type, size, degree of development and water content [35]. Ionizing radiation interacts with atoms and molecules to produce free radicals in cells. These radicals can damage the structure of biomolecules such as carbohydrates, lipids, proteins, enzymes and nucleic acids, affecting the primary metabolism of plants [36,37]. In addition, irradiation can affect biochemical processes such as photosynthesis, respiration, krebs cycle and the metabolism of biomolecules. According to Hasbullah et al. [38] and Hernández-Muñoz et al. [35], irradiation can also affect cell division and cause damage to chromosomes and DNA.

The increase in the number of shoots at a dose of 20 Gy could be associated with a hormetic effect. According to Calabrese [39], the hormetic effect is characterized by beneficial or stimulation of development at low doses; and toxicity, inhibition or death at high doses. According to Jalal et al. [40], reactive oxygen species (ROS) are associated with hormesis because they are signaling molecules that trigger different physiological, biochemical and molecular processes in plant development. In this study, doses higher than 60 Gy caused the death of the explants and a reduction in the number of shoots per explant, shoot length and number of leaves per shoot. The reduced development and increased mortality rate at high doses could be associated with a longer time exposure to 60Co. The high dosage of gamma ray causes production and accumulation of ROS, which are toxic to plant tissues [41,42]".

In this regard, Oliveira et al. [41] stated that the excess accumulation of free radicals resulting from water radiolysis produces negative effects on structural and functional biomolecules causing alterations in cellular metabolism, while Liu et al. [42] noted that high doses of gamma radiation can induce oxidative stress; this stress generates the formation of ROS that affects cell division and leads to apoptosis.

The mechanisms of the hormetic effect have yet to be fully elucidated; however, Iavicoli et al. [43] stated that hormesis is an adaptive preconditioning response to a stress of greater magnitude based on an evolutionary event. The hormetic effect has been observed in other in vitro mutagenesis studies with 60Co in golden-flowered vetchling (*Lathyrus chrysanthus* Boiss) [44], San Francisco lily (*Laelia autumnalis*) [16], shoreline purslane (*Sesuvium portulacastrum*) [45] and more recently in rice (*Oryza sativa* L.) [17]. These results suggest that gamma irradiation with 60Co could promote in vitro morphogenesis in explants in a dormant state in recalcitrant species.

The ISSR markers were able to detect somaclonal variations between individuals and the different doses of gamma irradiation evaluated. In this regard, Khan et al. [46] stated that ISSR markers produce multiple bands at the same locus, are highly reproducible and do not need prior information from the plant genome. In this study, primers UBC-808, UBC-836 and UBC-840 revealed the highest percentage of polymorphism and can be used for future analysis of somaclonal variation or genetic diversity in *V. planifolia*.

In general, individuals irradiated with doses of 100 Gy showed the least genetic similarity; however, for the rest of the doses evaluated, no clustering trend was observed. In vanilla, other studies involving somaclonal variation analysis using ISSR markers have observed that this species tends to be genetically unstable upon in vitro regeneration [29,31,47]. Ramírez-Mosqueda and Iglesias-Andreu [47] reported somaclonal variation during indirect organogenesis, with 71.66% polymorphism. Bello-Bello et al. [29] found an increase in the percentage of polymorphism with increasing concentrations of plant nanoparticles (AgNPs) in the culture medium during the growth of *V. planifolia*, with 25% polymorphism at a concentration of 200 mg·L−<sup>1</sup> AgNPs. Pastelín-Solano et al. [31] demonstrated that the number of subcultures during direct organogenesis is an important factor in the increase in somaclonal variation, obtaining % P greater than 15% from subculture number six. On the other hand, other studies found no somaclonal variation in *V. planifolia* [30,48–50]. Sreedhar et al. [49] did not observe somaclonal variation during long-term growth using ISSR and RAPD markers. Gantait et al. [48] did not observe somaclonal variation during direct organogenesis using ISSR markers. Ramírez-Mosqueda et al. [30] in variegated plants obtained during direct organogenesis in temporary immersion obtained 0% polymorphism using ISSR markers. Recently, Manokari et al. [50] through direct organogenesis demonstrated 0% polymorphism using markers based on start codon targeted (SCoT) polymorphism.

Somaclonal variation during in vitro culture can originate through various aspects such as: explant type, regeneration pathway, subculture number, culture duration, growth regulator type, genotype and ploidy level [22–51]. However, somaclonal variation can be induced by chemical and physical mutagenic agents. Gamma irradiation using 60Co can generate different types of mutations, namely deletions and insertions, translocations and base substitutions [52,53]. According to Jain [54], mutations produced by somaclonal variability are very similar to those produced spontaneously or by mutagenesis methods.

The somaclonal variation obtained in non-irradiated explants could be explained by the genetic nature of *V. planifolia*. In this regard, Nair and Ravindra [55], and Bory et al. [56] observed in vanilla somatic associations and anomalies in the number of chromosomes, being lower than the reported 2*n* = 32. This could explain why, during in vitro regeneration of vanilla, higher somaclonal variation is expected compared to other species that do not show somatic association or anomalies in the ploidy level. The somaclonal variation found in irradiated treatments, in addition to the genetic nature of the species, could be due to the high penetrating potential of gamma rays and mainly to the breaking of the chemical bonds in the DNA double strand, eliminating nucleotides or replacing them with new ones [14]. In this study, DNA mutations can probably affect homeotic genes with effects on the ability to regenerate new shoots.

Predieri [57] and Bairu et al. [51] state that in vitro culture increases the efficiency of mutagenic treatments due to the manipulation of explants in constant cell division under controlled conditions without biotic or abiotic factors that interfere with the mutagenic treatment. The effect of in vitro mutagenesis using 60Co to broaden genetic variation for breeding purposes has been studied in San Francisco lily (*Laelia autumnalis*) [16], rice (*Oryza sativa* L.) [17], ginger (*Zingiber officinale* Rosc.) [8], potato (*Solanum tuberosum* L.) [18], and tomato (*Lycopersicon esculentum* L.) [19].

The species *V. planifolia* has low genetic diversity due to the cuttings-based asexual reproduction [58]. This commercial propagation method limits the diversity of the species. In vanilla, somaclonal variation is an alternative to broaden the genetic base of this species and generate new alleles [31] that can address the inbreeding depression of this species. The increase in the genetic diversity of vanilla is an important factor contributing to tolerance to abiotic and resistance to biotic factors caused by different climate change scenarios to avoid its extinction. Gamma radiation is a very useful mutagenesis method to generate genetic variations for the improvement of this species. In addition, future studies are required to analyze flowering stage and ripe fruits with morphological and biochemical markers to find possible phenotype variation.

#### **5. Conclusions**

In this study, it was observed that gamma radiation has a hormetic effect on explants, promoting the formation of new shoots at low dose (20 Gy) and inhibition of sprouting and death at high doses (60–100 Gy). Furthermore, in vitro regeneration via direct organogenesis and the different doses of gamma irradiation evaluated with 60Co were shown to have an effect on somaclonal variation. The analysis of NJ clustering and Jaccard's genetic distance showed that the treatment without irradiation and the treatments with irradiation present genetic divergence from the donor plant. ISSR markers were shown to be efficient in detecting somaclonal variation. These results support the possibility of using gamma rays during in vitro culture to increase genetic diversity and undertake a vanilla breeding program.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae8060503/s1, Figure S1: Uncropped blot of Figure 3a, Figure S2: Uncropped blot of Figure 3b.

**Author Contributions:** Conceptualization, M.K.S.-F. and J.J.B.-B.; designed the experiments, analyzed the data, conducted data interpretation and drafted the manuscript, M.K.S.-F.; conducted all the experimental work, F.C.G.-M., S.C.-I., J.L.S.-C. and J.J.B.-B.; contributed to the conceptualization of the experiment and revising the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data in this study can be found in the manuscript or in the Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interests.
