Droplet vitrification: a lifeline for long-term conservation of threatened species Garcinia indica

Cryopreservation is a promising technique for the ex situ long-term conservation of plant biodiversity particularly of species that are not amenable to seed bank conservation. Garcinia indica (Thouars) Choisy is a species endemic to the biodiversity hotspot of the Western Ghats of India. Conservation of this species is difficult as it produces recalcitrant seeds, and therefore, it can only be conserved in the field genebanks or in their natural habitats. This high-value fruit tree species is listed as vulnerable due to the rapid loss of the natural population, and therefore, the conservation of its genetic diversity is imperative. This study described the first successful cryopreservation protocol for G. indica through a modified droplet vitrification technique using in vitro–derived shoots conserved in the In Vitro Gene Bank of the Indian Council of Agricultural Research - National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi. Among vitrification (V) and droplet vitrification (DV) techniques, only the DV technique yielded explant regeneration after cryopreservation. Apical shoots (2 mm long) collected from 24-wk-old explants (IC638183), without any pre-culture, showed maximum regeneration (51.76%) on Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzylaminopurine (BAP). The regenerated shoots were hardened in a mist chamber successfully (85% survival), which is essential for the future restoration of the species in the field. With genotypic-dependent variation, this protocol was applicable to the other three accessions (IC638184, IC638185, and IC638186) with an average of 43.7% regeneration after cryopreservation, which can be implemented for the effective conservation of G. indica germplasm.