Optimizing in vitro slow-growth conservation media for garlic under ambient conditions: further implications for core set accessions

Garlic is a bulbous crop exhibiting sufficient variations in morpho-biochemical characteristics. The entire garlic germplasm is conserved through clonal propagation at a field gene bank and thus prone to climatic changes’ effects. Therefore, maintaining the crop’s genetic diversity in a suitable alternative form is crucial. The current study standardized the protocol for slow-growth conservation of garlic (in vitro) at an ambient temperature. Plantlet regeneration through the shoot meristem was subjected to 21 treatments with three osmoticum, namely sucrose (1, 2, 3, and 4%), sorbitol (2 and 4%), and mannitol (2 and 4%), alone or in combination, including the control, at an ambient temperature in 2 × 21 factorial design. Furthermore, the identified treatment was validated using a core set of garlic accessions (46) to study the genetic response to in vitro slow-growth conservation. A pooled analysis revealed significant differences in the response of two garlic varieties, Bhima Omkar and Bhima Purple, to varying concentrations and combinations of three osmotic agents (p < 0.05). Over time, substantial shooting, rooting, and plant status changes were observed. However, treatment interaction effects showed no significant variation (P ≥ 0.05). In the first month of conservation, treatments with sucrose alone (1% sucrose, 2% sucrose, 3% sucrose, and 4% sucrose) recorded healthier and quicker growth, whereas those with sorbitol and mannitol alone resulted in slower growth. The same growth pattern was recorded in the third month of conservation, except that the survival rate of the plantlets decreased. Media in the sucrose-only treatments dried up sooner than those in the other media combinations. At 6 months, among the 21 medium combinations tested, Murashige and Skoog’s medium supplemented with 4% sucrose and 2% sorbitol demonstrated significantly favorable delayed growth under ambient temperature conditions (25 °C ± 2), achieving a 90–92% survival rate of plantlets, outperforming other treatments. A total of 46 garlic core set accessions were cultured in the specified medium. After 1 year of conservation with two subcultures at 6-monthintervals, growth parameters, including mortality (%), plant status, and shoot and root growth, were assessed. The plant status data revealed genotypic variability, grouping the accessions into three categories containing 19, 15, and 12 genotypes, respectively. These groups were ranked in descending order based on plant status, shoot and root growth, and mortality. Genetic purity analysis using three ILP primers indicated no significant molecular-level changes. This protocol presents an efficient, cost-effective, and scalable approach for the slow-growth conservation of diverse garlic genotypes using osmotic agents. Beyond minimizing natural deterioration during in vivo field gene bank, it also enables the identification of osmotic stress-tolerant genotypes, which hold promise as potential parental lines in breeding programs targeting drought resilience.