Embryo culture of coconut: The CPCRI protocol
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2007-02-08
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The field collection of embryos from mature nuts (9 months onwards), which can be easily identified for its hard eye and thick kernel, is done by means of a surface-sterilized cork borer. From the endosperm plug thus scooped out, the embryo is separated by using knife. The collected embryos are placed in distilled water. Once all the embryos are extracted, they are subjected to surface sterilization with 50% chlorine water for 20 minutes. The embryos are washed thoroughly in sterile water 4-5 times and inoculated individually into screw-cap bottles containing 2-5 ml sterile water (pH 5.7). The entire operation is to be done in surface-sterilized (absolute alcohol) inoculation hood (portable). The field-collected embryos are then transferred to Y3 solid medium within 2 months. The medium is supplemented with 30 g/1 sucrose and 1 g/1 charcoal for embryos of tall types and 60 g/ 1 sucrose and 2.5 g/1 charcoal for dwarf types. For the 8 months old nuts, embryos being small in size (1.5 to 4 mm), surface sterilization was done for 10 minutes. These embryos can be stored in half-strength Y3 medium for 1 month. Initially these embryos are inoculated in Y3 medium supplemented with 60 g/1 glucose and 2.5 g/1 charcoal. After 1 month, on attaining the full size, the embryo were transferred to regular retrieval medium. Cultures in the retrieval medium are initially kept in dark room till germination and later transferred to the illuminated room. Periodical sub-culturing was followed at monthly intervals. Once the plants attain the length of 5-7 cm, they were transferred to liquid medium. For rhizogenesis, the medium was supplemented with 1BA (5 ppm) and NAA (1 ppm). For pot establishment, sterilized sand, soil and coir dust in equal proportion were used as the potting mixture. The humidity was controlled in the initial stage of ex vitro establishment and macro nutrients were provided.
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Indian J.Horticulture 1999 v-56 i-4 p-348-353