Cryopreservation of Koovalam (Aegle marmelos L.) using vitrification technique (Record no. 289364)
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| 000 -LEADER | |
|---|---|
| fixed length control field | 05438nam a22001937a 4500 |
| 082 ## - DEWEY DECIMAL CLASSIFICATION NUMBER | |
| Classification number | 660.6 |
| Item number | PAR/CR PG |
| 100 ## - MAIN ENTRY--PERSONAL NAME | |
| Personal name | Parvathy, B |
| 245 ## - TITLE STATEMENT | |
| Title | Cryopreservation of Koovalam (Aegle marmelos L.) using vitrification technique |
| 260 ## - PUBLICATION, DISTRIBUTION, ETC. (IMPRINT) | |
| Place of publication, distribution, etc | Vellayani |
| Name of publisher, distributor, etc | Department of Plant Biotechnology, College of Agriculture |
| Date of publication, distribution, etc | 2021 |
| 300 ## - PHYSICAL DESCRIPTION | |
| Extent | 71p. |
| 502 ## - DISSERTATION NOTE | |
| Dissertation note | BSc- MSc Int. |
| 520 ## - SUMMARY, ETC. | |
| Abstract | 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. |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Plant Biotechnology |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Cryopreservation |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Koovalam |
| 650 ## - SUBJECT ADDED ENTRY--TOPICAL TERM | |
| Topical term or geographic name as entry element | Aegle marmelos L. |
| 700 ## - ADDED ENTRY--PERSONAL NAME | |
| Personal name | Deepa S Nair (Guide) |
| 942 ## - ADDED ENTRY ELEMENTS (KOHA) | |
| Source of classification or shelving scheme | |
| Item type | Theses |
| Not for loan | Collection code | Permanent location | Current location | Shelving location | Date acquired | Full call number | Barcode | Date last seen | Koha item type |
|---|---|---|---|---|---|---|---|---|---|
| Reference Book | KAU Central Library, Thrissur | KAU Central Library, Thrissur | Theses | 2022-04-23 | 660.6 PAR/CR PG | 175304 | 2022-04-23 | Theses |