PubMed · 11658704
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Paul Bagne. 1983. High-tech breeding.. https://pubmed.ncbi.nlm.nih.gov/11658704/
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Marine diplonemids (Diplonemidae and Hemistasiidae), once considered difficult to culture, can now be introduced into stable axenic cultures and grown to high densities. This chapter provides detailed protocols on establishing clonal cultures from environmental cells, their axenization and routine maintenance, as well as instructions on mid- and large-scale cultivation, estimation of cell density, and cryopreservation of selected species.
Experiments were initiated to develop protocols for four species of Dioscorea, namely D. bulbifera L., D. oppositifolia L., D. alata L. and D. cayenensis Lam., and to assess the effects of various factors on the survival and regrowth of explants after rewarming. The treatments consisted of three different methods-vitrification, droplet and a modified droplet method-as well as the following variables-cryoprotective solution, sucrose concentration in the preadaptation medium and cold acclimation. While most of the protocols resulted in low to zero survival and regrowth rates, maximum survival rates of 100% and 75% were obtained for D. oppositifolia L. and D. cayenensis Lam., respectively, using two different protocols of the modified droplet method. A higher average survival rate was obtained using the droplet and modified droplet techniques than for the original vitrification methods. When the average of all the experiments was taken, more than one-half (52%) of the surviving explants developed further within 1 month with the modified droplet method compared to zero regrowth with the two other methods. The optimum protocol for cryopreservation is specific for each genotype.
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