Vitrification of excised embryos of jackfruit (Artocarpus heterophyllus lamk.)

Cryopreservation using the vitrification method involves treatment of tissues with a concentrated vitrification cocktail, which forms an amorphous glass without crystallizaton during rapid cooling in liquid nitrogen. During the process, treatment with loading solutions with cryoprotective properties is reported to reduce the harmful effects of the vitrification solution that emanate from osmotic stress and chemical toxicity. The effect of various loading solutions was studied in a preliminary experiment, wherein the embryos were treated with 6 different loading solutions, followed by exposure to Plant virification Solution 2 (PVS2) for 30 minutes before plunging into liquid nitrogen. The results showed that without freezing, all the loading solutions did not have deleterious effects on the jackfruit embryos as high survival rates of 81-96% were obtained. On freezing, the non-loaded embryos (control) did not survive virification, suggesting that loading was necessary to enhance the viability and survival of jackfruit embryos. The frozen embryos loaded with 25% PVS2 gave the highest viability (43.3%) and was thus selected as the best loading solution for jackfruit embryos for the subsequent experiments. In order to determine the optimum time of exposure to the best loading solution, the embryos were loaded with 25% PVS2 for 0, 8, 12, 14, 16, 18and 20 hours, followed by exposure to PVS2 for 30 minutes before freezing. The non-frozen embryos maintained 100% viability and 96.7% survival up to 20 hours of exposure. On freezing, 12, 14 and 16 hours of loading were equally advantageous, but 14 hours showed the highest with 18.3% viability and 13.3% survival rates. This was then chosen as the optimal time of exposure for the next experiment. Longer exposures of 18 and 20 hours were excessive and decreased the percentage viability and survival to less than 7%. Successful vitrification requires the use of a highly concentrated yet relatively non-toxic vitrification cocktail. Therefore the third experiment assessed the effects of different vitrification solutions. Loaded embryos were exposed to 5 different vitrification solutions for 30 minutes before plunging into liquid nitrogen. In the absence of freezing, 100% viability and 96-100% survival rates were obtained for all the vitrification solutions evaluated, suggesting that they were relatively non-toxic to the embryos within the exposure time. On freezing, embryos treated with L Solution gave the highest viability (30%) and survival (24%), which were significantly better than PVS2 and Watanabe Solution. Embryos treated with PVS and PVS3 did not survive vitrification, suggesting that vitrification solutins are species-specific. Jackfruit embryos were treated with L Solution for 0, 15, 30, 45, 60, 75, 90, 105 and 120 minutes before freezing. Without freezing, high viability (94.6-100%) was obtained up to 120 minutes exposure. Following freezing, the effects of L Solution showed a time dependent viability. The percentage viability increased with time and reached an optimum of 47.4% after 75 minutes before declining to only 6.8% after 120 minutes of exposure. It is thus concluded that loading with 25% PVS2 for 14 hours, followed by exposure to L Solution for 75 minutes was optimal for the vitrification of jackfruit embryos. Viability and survival rates of 47.4% and 35.4% can be obtained.