1. Introduction
Garlic (
Allium sativum L.) is a vegetable native to Central Asia and is one of the most important species of the
Allium genus due to its particular taste and smell, as well as its beneficial properties for human health [
1]. Garlic bulbs contain organosulforates, flavanols, saponins, and sapogenins—substances with great potential in medicine and agriculture [
2,
3]. These compounds have antimicrobial, antioxidant, and flavor-enhancer effects, among others [
4].
There are 910 varieties of garlic grown in 35 countries [
5], but some of these are only conserved under field conditions and, therefore, are at risk of loss due to biotic and abiotic factors, even during post-harvest [
6]. For that reason, it is important to carry out garlic preservation and its genetic resources [
7].
Germplasm banks are essential to preserve the genetic diversity of species of agri-food importance, such as garlic. There are 1750 germplasm banks around the world that preserve different species in medium-term and long-term conservation, including elite and local varieties and wild relatives [
8]. The preservation of genetic resources is stipulated in international treaties such as the Convention on Biological Diversity and the International Treaty on Plant Genetic Resources for Food and Agriculture [
9].
Conservation of sub-orthodox and recalcitrant seeds and vegetatively propagated species, such as garlic, is carried out by in vitro culture techniques [
10]. In vitro plant culture enables cells, tissues, and organs to grow in aseptic culture media [
11].
Minimal growth is an in vitro culture technique for medium-term plant conservation, from a few months to more than a year, according to the species [
12]. Plant growth is slowed by chemical and physical factors. Some of these factors are the decrease of culture medium nutrients, the addition of osmotic agents, growth retardants, mineral oils, low conservation temperature, low light intensity, and shorter photoperiod [
13].
This medium-term plant conservation technique has been applied to different species using specific protocols for each one and even for each genotype due to genetic diversity [
14]. Regarding garlic, Benke et al. [
15] reported 70% survival of the Bhima variety after five months in Murashige and Skoog (MS) culture medium supplemented with sucrose (68.46 g L
−1) and sorbitol (36.43 g L
−1). Likewise, Pardo et al. [
16] obtained 73% survival of the Boconó clone after seven months of storage in MS medium (25%) containing 45 g L
−1 of sucrose. Both investigations were performed under standard light and temperature conditions (16 h light photoperiod, 25 μm m
−2 s
−1, and 25 ± 1 °C). On the other hand, Hassan et al. [
17] preserved Balady and Seds 40 varieties for 15 months at 4 °C in MS medium with sorbitol (72.87 g L
−1) in dark conditions and obtained survival rates of 35.7% and 90% for each variety, respectively.
Different garlic varieties and their wild relatives are in medium-term and long-term preservation worldwide [
18]. However, in most cases, there is a cultivar- or genotype-specific response. In Mexico, there are garlic varieties of agri-food and breeding importance for which there are no in vitro conservation protocols, and they are not stored in a germplasm bank. In this sense, it is vital to generate in vitro conservation protocols because the current conservation of garlic germplasm depends on field regeneration and noncontrolled storage conditions where it is exposed to biotic and abiotic threats, such as unfavorable environmental conditions, pillage, pests, and diseases. Minimal growth provides a medium-term in vitro conservation alternative that allows to safely preserve important germplasm under controlled conditions, efficiently using space and resources. Due to this, the objective of this work was the in vitro preservation of three Mexican garlic varieties by minimal growth.
3. Discussion
Preservation of plant genetic resources is essential to guarantee the well-being of the world’s population with increasing agri-food needs [
21]. Also, plant genetic resource conservation contributes to ensuring agricultural and food security for many generations, either, through the use of these resources, to restore specific plant populations or to perform genetic improvement and generate varieties better adapted to the challenges ahead [
22].
In this sense, genetic diversity preserved in gene banks must be immediately available [
23]. Therefore, minimal growth is an in vitro conservation technique that facilitates the availability, accessibility, and multiplication of plant germplasm [
18].
In this study, 5 °C allowed the plant’s conservation time to reach up to 365 days in the Pebeco, Tacátzcuaro Especial, and Huerteño varieties. These results are the first report of a medium-term conservation protocol for these varieties. Cold is one of the main factors that slows plant growth and development [
24]. Low temperatures reduce the photosynthetic rate; however, in some cold-sensitive species, they cause depolymerization of chloroplast microtubules [
25].
The conservation period of these three varieties in the present study was longer than reported by Benke et al. [
15] who preserved the garlic variety Bhima for five months and obtained similar survival with the same culture medium (M5) under standard incubation conditions. Although the M5 culture medium formulated with basal MS medium, sucrose, and sorbitol did not limit the growth of the explants, it favored their survival.
On the other hand, sucrose, sorbitol, and sugars, in general, at high concentrations can cause osmotic stress in plant cells [
26]. Osmotic stress affects cell division, morphogenesis, and survival [
27]. Gelmesa et al. [
28] reported that 0.2 M sorbitol reduced in vitro growth to different degrees in three genotypes of potato (
Solanum tuberosum L.). In this study, most garlic plants formed bulbs at the three conservation temperatures and mainly at M1 culture medium (100 g L
−1 sucrose), probably due to a plant survival mechanism strategy induced by osmotic stress. For instance, Alexopoulos and colleagues [
29] found that a higher concentration of 8% sucrose in the MS culture medium contributed to enhanced in vitro bulb production in sea lilies (
Pancratium maritimum). Similarly, in a separate study, the inclusion of 7% sucrose in the MS culture medium was shown to promote bulb formation in elephant garlic (
Allium ampeloprasum L.) [
30]. Moreover, in another investigation conducted by Youssef et al. [
31], the use of 9% and 12% sucrose in the MS culture medium resulted in a notable increase in bulb formation for lilies (
Lilium orientalis cv. “Starfighter”).
Other factors that influence growth and survival during in vitro culture are the explant type, its phenological state, the size and volume of the culture container, and the volume of culture media used [
32]. In this sense, during medium-term in vitro conservation, one of the most important variables is shoot height since it is one of the variables that allows choosing the best conditions to extend subculture time [
33]. In this study, garlic plants showed their maximum growth in a relatively short period at 25 °C compared with other cultivated species like sweet potato and native species like
Alcantarea nahoumii (Bromeliaceae) (Leme) J. R. Grant, which were conserved for 4 and even 24 months without significant growth, respectively [
34,
35]. Nevertheless, shoot growth did stabilize in time, which indicates that all variables and their effects on variety conservation should be considered and evaluated during an appropriate period. Although low temperatures did not limit initial rapid plant growth, they did extend subculture time with higher survival rates in all cases with better morphological characteristics.
Another aspect to take into account when establishing a conservation protocol such as minimal growth is the effect of genotype under specific in vitro conditions. In the present study, different responses can be observed between the three garlic varieties evaluated, whose survival ranges oscillate between 95.8 and 54.2%. This coincides with what was found by Benke et al. [
15] who obtained survival percentages between 20 and 70 depending on the garlic genotype evaluated. Therefore, it is necessary to perform the necessary research in order to generate species-specific and sometimes, as in the case of garlic, variety-specific conservation protocols.
One of the main obstacles in in vitro culture is endogenous contamination of the explant [
36]. Bacterial growth in the initial stages of in vitro culture is frequent; however, bacterial contamination has also been observed during the multiplication and acclimatization stages [
37].
In this study, more bacterial contamination was observed at 18 °C in culture medium M2 (MS medium at 25% supplemented with 45 g L
−1 sucrose). This could be attributed to the fact that the optimum temperature range for the development of the garlic plant is 12 to 24 °C, and this could also benefit the growth of the natural microbiota of garlic [
38].
Regarding culture media, sucrose is the main carbon source in plants in vitro [
39]. In case of minimal growth, over time the carbon source is gradually reduced in the culture medium, and the remaining components in it concentrate due to medium dehydration caused by water evaporation. These phenomena cause greater plant stress due to the eventual change in culture medium components availability and osmotic potential. Osmotic stress could be the reason for a greater presence of endogenous garlic microbiota over time. Also in bacteria, sucrose operons are expressed in the presence of sucrose and when other preferred carbon sources are depleted [
40].
On the other hand, endophytic microorganisms colonize the vascular tissues of plants and, in most cases, survive surface chemical disinfection [
41] and, therefore, can appear months after in vitro establishment, as is the case in the present study. These microorganisms can have a negative or positive effect on plants. In this sense, a bacteriostatic effect has been observed in endophytes isolated from garlic bulbs [
42], and endophytic bacteria from garlic roots promote plant growth in vitro [
43]. Furthermore, garlic bulbs and garlic oil have different components with antifungal effects [
3]. This could be one of the reasons why only major bacterial contamination was observed in two of the three varieties evaluated. Likewise, this could favor the survival of contaminated explants. However, contaminated living plants were discarded for evaluation purposes, and after 365 days of evaluation, they were preserved by minimal growth.
In the present study, plant regeneration after 365 days in conservation by minimal growth was lower than plant survival for all three garlic varieties. Nevertheless, Tacátzcuaro Especial has the highest number of regenerated plants. These results are in accordance with other minimal growth reports in which lower explant regeneration than explant survival at the end of the conservation period was obtained [
44,
45]. This suggests that plant survival is not sufficient to choose an adequate number of initial plants for medium-term conservation.
The capacity of plant cells to regrow after a medium or long conservation period depends on many factors, like the establishment of adequate regrowth conditions, species, the status of the donor plant or initial material, explant type, and the in vitro conservation technique [
22].
5. Conclusions
In the present study, optimal conditions for minimal growth conservation of Pebeco, Tacátzcuaro Especial and Huerteño garlic varieties for a one-year period were established. Basal MS culture medium supplemented with sucrose and sorbitol at 5 °C led to the best response for the conservation of three Mexican garlic varieties. With these results, a new alternative was generated for the in vitro preservation of important garlic varieties that did not have a safe and sustainable conservation strategy under controlled conditions.
Despite the number of regenerated plants obtained after a year of minimal growth conservation, based on the survival results, it can be recommended to reduce the storage time from 365 to 180 days in the case of Pebeco and Huerteño and to 270 days for Tacátzcuaro Especial. This strategy will guarantee higher numbers of regenerated plants.
Furthermore, endogenous contamination occurred in Pebeco, Tacátzcuaro Especial, and Huerteño varieties, so it is recommended to carry out additional disinfection treatments during the in vitro process and to isolate and identify garlic endophytic microorganisms to provide valuable information on its potential application. Regardless of contamination rates, regenerated aseptic plants are sufficient as starting material for a future micropropagation step. Results obtained during this study help to choose an adequate number of plants for medium-term conservation in future experiments with different varieties.
Even though this study was carried out with specific Mexican garlic varieties, the resulting conservation alternative can be transferred to other varieties generated from the same commercial variety “Taiwan”; therefore, its application can be considered global.