Cryoconservation of koovalam (Aegle marmelos L.Corr.) by encapsulation-dehydration technique

Abstract

The study entitled “Cryoconservation of Koovalam (Aegle marmelos L. Corr.) by encapsulation-dehydration technique,” was carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2015-2017. The objectives of the present study were to standardize a cryopreservation protocol for Aegle marmelos using encapsulation and dehydration technique and to assess the genetic fidelity of plantlets regenerated from encapsulated axillary buds after cryostorage, using molecular markers.

Investigation was carried out in three phases viz., enhancement of multiplication rate, standardization of in vitro conservation using the encapsulation-dehydration technique and assessment of genetic fidelity of the regenerated plantlets using ISSR markers. Single node segments with axillary buds from in vitro maintained cultures were used as the explants in all the experiments. Among the twelve combinations of auxins and cytokinins were tried, MS (Murashige and Skoog, 1962) medium supplemented with BA 2 mg L-1 + IBA 0.5 mg L-1 was found to be the best treatment with respect to shoot multiplication (9.33 shoots per explant). The additives chitosan (CH), thidiazuron (TDZ) and adenine sulphate (AdS) at different concentrations were supplemented in two different media i.e. 1) hormone free MS medium and 2) MS + BA 2 mg L-1 + IBA 0.5 mg L-1 to study their effect on shoot proliferation. The best shoot proliferation response obtained for each additives were MS + BA 2 mg L-1 + IBA 0.5 mg L-1 + chitosan 10 mg L-1 (35.66 shoots per explant), MS + AdS 60mg L-1 (5.33 shoots per explant) and MS + BA 2 mg L-1 + IBA 0.5 mg L-1 + TDZ 0.02 mg L-1 (20.00 shoots per explant). Even though higher shoot proliferation was observed in CH and TDZ supplemented media, they exhibited morphological abnormalities. Normal shoots were obtained with AdS supplemented medium, but shoot proliferation was less compared to MS + BA 2 mg L-1 + IBA 0.5 mg L-1. Hence MS + BA 2 mg L-1 + IBA 0.5 mg L-1 was used as basal medium for cryopreservation studies. Encapsulation-dehydration technique of cryopreservation involved various steps like preconditioning, encapsulation, pre-culture, dehydration (desiccation), thawing and recovery. Nodal segments with single axillary buds were used as the explant. In preconditioning experiment, PC2 (sucrose 0.1M in semi solid MS for 7 days) was selected as the best preconditioning treatment, which produced maximum shoot proliferation (5.50 shoots per culture) when cultured on basal medium. Among different encapsulation treatments, maximum shoot proliferation of (6.66 shoots per culture) was obtained in the beads formed with sodium alginate 3.5 per cent in modified MS medium and calcium chloride 100 mM, when cultured on modified basal medium (½ MS + BA 2 mg L-1 + IBA 0.5 mg L-1). Pre-culture experiments were conducted using preconditioned and encapsulated explants. The pre-culture treatment selected was liquid MS medium supplemented with sucrose 0.5 M and DMSO 3 per cent for 3 days, which gave maximum shoot proliferation (3.66 shoots per explant) in modified basal medium. The preconditioned, encapsulated and pre-cultured beads were subjected to 0 to 7 h of desiccation under laminar airflow. The moisture content declined from 82 per cent to 12.60 percent on 7 h of desiccation. The desiccated beads were then cryopreserved in liquid nitrogen for 2h, followed by thawing for 30-60s at 40oC and inoculated on to recovery medium ½ MS + BA 2 mg L-1 + IBA 0.5 mg L-1. Survival (66.67 per cent) and regeneration (50 per cent) could be obtained only at 6h of desiccation when the moisture content was 19.50 per cent. The beads when stored in liquid nitrogen for different duration did not show any significant variation with respect to survival and regeneration. The genetic fidelity of plantlets regenerated from encapsulated axillary buds subjected to cryostorage were analysed using ISSR markers. Among the nine primers tried, four primers UBC 807 (AGAGAGAGAGAGAGAGT), UBC 840 (GAG AGA GAG AGA GAG AYT), UBC 847 (CACACACACACACACART) and UBC 826 (ACACACACACACACACC) that gave scorable (5-7) bands were selected for the study. The ISSR banding patterns of the cryoregenerated plantlets and control plants were identical, which indicated the genetic stability. This study was successful in developing a protocol for cryopreservation using encapsulation-dehydration technique in A. marmelos with 50 per cent regeneration efficiency.