BSc- MSc Int.

The research work entitled “Cryopreservation of Koovalam (Aegle marmelos
L.) using vitrification technique” was conducted in the Department of Plant
Biotechnology, College of Agriculture, Vellayani during the year 2020-2021. The
objective of this study was to determine the effect of vitrification technique of
cryopreservation on recovery and regeneration of A. marmelos plantlets and assessment
of their genetic fidelity using molecular markers.
In vitro cultures of A. marmelos were established via embryo culture and the
nodal segments containing a single axillary bud were inoculated on MS medium
supplemented with BA 2 mg L-1
and IBA 0.5 mg L-1
to enhance the release of axillary
buds. The axillary buds obtained from these in vitro shoots were used as explant for the
study. In vitro conservation of A. marmelos was attempted using the vitrification
method of cryopreservation. Two vitrification techniques viz., simple vitrification and
encapsulation-vitrification were tried. The main steps involved in vitrification protocol
are preconditioning, encapsulation (in case of encapsulation vitrification), pre-culture,
loading, vitrification, cryostorage, thawing, unloading and recovery.
Nodal segments (ca. 5-8 mm) bearing single axillary buds were subjected to
preconditioning in MS medium containing sucrose 0.1 M for 7 days. The
preconditioned explants were encapsulated (in the case of encapsulation vitrification)
using sodium alginate 3.5 % and CaCl2 100 mM. The preconditioned explants with and
without encapsulation were subjected to pre-culturing in liquid MS medium containing
DMSO 3 % and sucrose 0.5 M for 5 days at 4 ºC under dark conditions with medium
change after every 24 h.
The pre-treated explants were then transferred to a sterile cryovial containing
autoclaved loading solution (liquid MS with glycerol 2 M and 0.4 M sucrose) and
incubated aseptically for 20 min. Post incubation, the explants were treated with PVS2
or PVS3 solutions for different durations ranging from 0 to 210 min with 30 min
interval. The vitrified explants were then plunged directly into liquid nitrogen and
stored for 2 h. Following cryo-treatment, the explants were warmed in a water bath
68
maintained at 40 ºC for 2 min. The warmed explants were treated with unloading
solution (liquid MS with sucrose 1.2 M) for 20 min and then, inoculated in regeneration
medium (Half MS supplemented with BA 2 mg L-1
and IBA 0.5 mg L-1
).
Among the various treatments in both the techniques, non-encapsulated
explants subjected to PVS2 treatment for 60 min alone survived. The survival and
regeneration obtained were 65.57±1.57 % and 61.10±4.16 % respectively. The new bud
initiation was observed in 28.33±1.02 days of inoculation in the regeneration medium.
The cryo-recovered explants produced 2.09±0.44 shoots per explant, 1.72±0.10 nodes
per shoot and 4.17±0.08 cm long shoots. Explants treated with PVS3 solution turned
albino and did not show any signs of survival. The treatments in encapsulationvitrification techniques also did not show any sign of survival until eight weeks of
inoculation in the recovery medium.
Axillary buds obtained from single seeds, subjected to simple vitrification
method of cryopreservation using PVS2 solution for 60 min were cryostored for 2 h, 1
day, 1 week, 2 weeks and 1 month in liquid nitrogen. No significant difference was
observed in terms of survival per cent, regeneration per cent, days to bud initiation,
shoots per explant, shoot length and nodes per shoot for various durations of
cryostorage.
The genetic stability of cryo-regenerated plants was determined using ISSR
markers. Out of the eight primers screened, five primers viz., UBC-809
(AGAGAGAGAGAGAGAGG), UBC-819 (GTGTGTGTGTGTGTGTA), UBC-826
(ACACACACACACACACC), UBC-836 (AGAGAGAGAGAGAGAGYA) and
UBC-840 (GAGAGAGAGAGAGAGAYT) gave clear distinct bands. The banding
profile of control plants and cryo-regenerated plants were identical indicating that the
regenerated plants were true to type.
The simple vitrification technique standardised for the cryopreservation of A.
marmelos using axillary bud as explant encompasses preconditioning in MS medium
supplemented with sucrose 0.1 M for 7 days, pre-culture in liquid MS medium
supplemented with DMSO 3 % and sucrose 0.5 M for 5 days, loading treatment (liquid
69
MS supplemented with glycerol 2 M and sucrose 0.4 M) for 20 min, vitrification with
PVS2 solution for 60 min, cryostorage in liquid nitrogen, thawing at 40 o C for 2 min,
unloading treatment (liquid MS supplemented with sucrose 1.2 M) for 20 min and
inoculation in regeneration medium (Half MS supplemented with BA 2 mg L-1
and
IBA 0.5 mg L-1
). The standardised protocol resulted in 65.57 % survival and 61.10 %
regeneration.