56 MARCH 2022 • WORLD AQUACULTURE • WWW.WA S .ORG 37, a population of granular cells was seen along with agranular cells. Regular monitoring revealed that the number of granular cells gradually increased (Figs. 8 and 9) or converted from agranular cells to granular cells. On day 54, aragonite micro-crystals were seen on culture plates. At the same time, in the second batch (A2), 1 × 3 mm size of mantle pieces folded with a shell particle of about 0.6 mm were inserted in about 6 ×15 mm rectangular pocket of mantle tissue. Mantle tissues were maintained in nutrient agar in culture flasks (Fig. 10), a culture medium in which supplementary substances were added in stages. Drops of liquid from flasks were separately analyzed periodically to monitor bacterial and other growth. On day 92, an irregular pearl sac started appearing within the mantle tissue, as observed from the bottom of two culture flasks during monitoring without opening the flasks (Fig. 11). Microscopic analysis indicated a wide spectrum of colors over the surface of irregular pearls formed, and high luster indicated that the pearl sac contained predominately aragonite crystals. Specimens of marine oysters Pinctada margaritifera ranging from 18 months to 2 years in age were selected and prepared (Figs. 5 and 6). Mantle tissues were surgically removed and sterilized with strict aseptic protocols using filtered seawater, ethanol and antibiotics. These tissues were transported from the Andaman Islands to a tissue culture and cell biology facility located in Prayagraj, Uttar Pradesh, about 2,000 km away. Throughout transport, suitable osmolarity and alkalinity were maintained in the culture medium. Tissues were healthy, even after 72 h in the culture medium. Tissues were again sterilized and cut into different sizes (Fig. 7) to prepare explants for a series of batch experiments. In the first batch (A1), tissues were placed on culture plates and culture medium and other supplementary minerals were added, as well as micronutrients and proteins extracted from various sources of marine origin such as seaweed, selected species of algae, calciferous substances of pearls/shells, etc. Microscopic analysis identified cell proliferation on day 6. Mixed cells were observed spreading from explants in prismatic structures. On day FIGURE 5. Marine oysters selected for tissue culture operations. FIGURE 4. A collection of black pearls produced in P. margaritifera. FIGURE 7. Pieces of mantle are cut for explants. FIGURE 6. Mantle extraction from P. margaritifera. FIGURE 8. Granular cell proliferation in the explant.
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