Conservation protocols for Ensete glaucum, a crop wild relative of banana, using plant tissue culture and cryopreservation techniques on seeds and zygotic embryos

Abstract

Ensets (Ensete spp., family Musaceae), are multipurpose plants with great versatility and potential as a food security crop, although they are cultivated only in Ethiopia and few other regions of Africa. Currently due to loss of habitat, intensification of agriculture, genetic diversity of both wild and domestic ensets is at high risk of erosion. Ensete glaucum (Roxb.) Cheesman, a less-known and under-utilized crop wild relative of banana, is found as natural strands in North-eastern regions of India. No report exists on seed conservation or cryopreservation of E. glaucum and the present work was undertaken to fill this knowledge gap. Mature fruits of two seeded accessions of E. glaucum (MZU-DBT-T15 and HSB-8) were collected from plants in natural populations from two geographically distinct North-eastern region of India. Seed morphology, moisture content (MC) of seed and embryo, viability and germination of excised embryos, desiccation and cryosensitivity were studied. Fresh seed (31% MC) viability measured through in vitro germination of excised embryos was not significantly different in both accessions (87–89%). Seeds could tolerate desiccation up to 5–10% MC without significant loss in germination potential (83–84%) and safely stored up to 1 year at room temperature (RT), without loss of viability. Air dehydrated seeds (10 ± 2% MC) and embryos (12 ± 2% MC) were cryopreserved (in liquid and vapour phase of liquid nitrogen). High post-thaw recovery was achieved in both tissues i.e. seeds (80.2 ± 2.2%) and embryos (83.1 ± 2.6%) by in vitro culture of excised embryos on MS medium + 2 µM gibberellic acid + 1 µM 6-benzyl amino purine (BAP) + 1 µM ascorbic acid. The seedlings converted into multiple shoots (15–20 shoots/explant) within 2 months of culture on medium with MS salts + 10 µM BAP + 1 µM indole-3-acetic acid (IAA) + 1 µM ascorbic acid. This study is the first successful report of cryopreservation of E. glaucum using simple air dehydration. Using the method devised, seeds and zygotic embryos of the two accessions of E. glaucum are safely conserved in the Cryogenebank, while in vitro plantlets are maintained in the In Vitro Genebank of ICAR-NBPGR, New Delhi, India. This study is the first successful report of cryopreservation of Ensete glaucum using seeds and zygotic embryos