**3.3 Somatic embryogenesis and plant regeneration**

In black pepper primary embryogenic cultures can be established as per the method described by Nair and Dutta Gupta (2003). Culture the surface sterilized seeds on agar gelled full-strength, PGR-free SH (Schenk and Hildebrandt, 1972) medium containing 3.0% (W/V) sucrose under darkness. Primary somatic embryos (PEs) derived from micropylar tissues of germinating seeds after 90 days could be utilized for inducing secondary somatic embryogenic cultures.

Primary somatic embryo clumps having pre-globular to torpedo shaped embryos (5–6 visible embryos per seed) were carefully detached and inoculated on half strength PGR-free SH medium containing 1.5 % sucrose and gelled with 0.8% agar (Bacteriological grade, Himedia). The pH of the medium was adjusted to 5.9 prior to autoclaving. Cultures were maintained at darkness at a temperature of 25±2oC. The culture conditions remained the same for all further experiments unless otherwise specified. While inoculating, the PEs were uniformly spread on the surface of the medium. Secondary embryogenic cultures were further maintained by subculturing on SH medium containing 1.5% sucrose at intervals of 20 d. The proliferating SEs were spread periodically on the surface of the medium, to facilitate proliferation.
