Development of Cryopreservation Protocol for Aquilaria malaccensis Lam., a Recalcitrant Seeded Tropical Tree Species.

BACKGROUND: Cryopreservation is the only possible process for long-term germplasm storage of species with recalcitrant behavior. Aquilaria malaccensis is a recalcitrant seeded tropical tree that produces a distinctive fragrance which has high value in the commercial market. OBJECTIVES: In the present study, we aimed to develop possible long-term storage techniques for A. malaccensis germplasm. MATERIALS AND METHODS: We used zygotic embryos and in vitro derived nodal buds as explants. The experiments were performed based on dehydration and encapsulation-dehydration methods. RESULTS: When the dehydration technique was applied, survival (43%) and regeneration (23%) was found to be higher for zygotic embryos than for in vitro derived nodal buds (13% & 10% respectively). In addition to moisture content within the tissue during cryogen exposure, dehydration duration has an important contributory role in post cryo-survival and regeneration. A slight increase in survival (47%) and regeneration (30%) were observed in zygotic embryos with a modification to rehydration after re-warming, while such a change did not improve success with in vitro derived nodal buds. In contrast, the encapsulation-dehydration technique was found to be more effective for in vitro derived nodal buds than zygotic embryos. All the encapsulated nodal buds that survived (27%) regenerated into plantlets while encapsulated zygotic embryos failed to regenerate into plantlets. CONCLUSION: This is the first report on the cryopreservation of A. malaccensis and the developed protocol conveys a comprehensive idea of its reliability for the long-term storage of this desiccation sensitive (recalcitrant) seeded tree species.

A comparative study of vitrification and encapsulation-vitrification for cryopreservation of protocorms of Cymbidium eburneum L., a threatened and vulnerable orchid of India.

Cryopreservation of the threatened orchid Cymbidium eburneum L. was successfully achieved using encapsulation-vitrification and vitrification. Comparing the two methods tested, it was observed that regeneration of protocorms cryopreserved using encapsulation-vitrification was higher than with vitrification. To achieve optimal regrowth after cryopreservation, protocorms were precultured for 24 h with 0.2 M sucrose for vitrification and with 0.7 M sucrose for encapsulation-vitrification, reaching 60 percent and 70 percent regeneration, respectively. With both techniques employed, a 20 min exposure duration to Plant Vitrification Solution 2 (PVS2) led to optimal regeneration after cryopreservation. A maximum of 66 percent regeneration was achieved after cryopreservation using encapsulation-vitrification, whereas it was only 50 percent after cryopreservation using vitrification. The same regeneration pattern was observed with protocorms cryopreserved using both techniques employed. This is the first report of long-term conservation by cryopreservation of C. eburneum protocorms.

Vitrification-based cryopreservation of shoot-tips of Pinus kesiya Royle ex. Gord.

The present investigation was aimed at developing a protocol for long-term preservation of germplasm of Pinus kesiya Royle ex. Gord. through vitrification. Some of the critical components affecting explant tolerance to cryopreservation, such as effects of preculture, vitrification solutions, exposure time to vitrification solutions, volume of vitrification solution and its toxicity, washing of vitrified tissues after thawing, were analysed. The results showed that shoot regrowth of P. kesiya shoot-tips was considerably affected when exposed to cryoprotectants for longer periods of time (longer than 10 min). Among different vitrification solutions studied, maximum survival (76 percent) of shoot-tips was achieved with mVSL (using 0.6 ml of the solution) in MS basal medium containing 4.0 mg l-1 N6-benzyladenine (BA).

Long-term conservation through cryopreservation of immature seed of Mantisia spathulata and Mantisia wengeri; two endangered plants of north-east India.

A successful protocol for long-term conservation of two endangered plants viz. Mantisia spathulata and M. wengeri has been devised through cryopreservation of immature seeds. Immature seeds of both the species were precultured in 0.6 M Sucrose and 2 M Glycerol for 3 h at 24 ± 2 degree C. Precultured seeds were then desiccated under the airflow of 27 ± 3 m min -1 velocity inside laminar air flow cabinet for different time periods. The seeds were then cryostored in liquid nitrogen for an hour. A maximum of 40 percent and 36.6 percent seed germination was recorded after cryostorage at moisture contents of 26.0 percent and 16.2 percent for M. spathulata and M. wengeri respectively. To protect these rare plants against loss due to disease, insect damage, or natural disaster a back up collection has been established using the protocol and applied to a large number of immature seeds that were obtained from the ex situ plants growing in the experimental garden of the North-eastern Hill University, Shillong.