Method for regeneration of coniferous plants by somatic embryogenesis

A method for reproducing plants selected from the group consisting of Pinus taeda, Pinus rigida, Pinus serotina, Pinus elliottii, Pinus palustris, and hybrids thereof, by somatic embryogenesis which comprises:

• (a) placing a suitable explant selected from the group consisting of immature zygotic embryos and megagametophytes containing immature zygotic embryos on culture initiation medium containing a sufficient amount of nutrients, 0.1 to 5.0 mg/l of auxin, 0.1 to 1.0 mg/l of cytokinin, 10.0 to 40.0 g/l of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and a level of gelling agent selected from the group consisting of 2.5 to 4.5 g/l of agar, 0.5 to 1.5 g/l of gellan gum, 3.0 to 5.0 g/l of agarose, and 1.5 to 3.0 g/l of AGARGEL, for 2 to 14 weeks under suitable environmental conditions to grow a culture containing embryogenic tissue;
• (b) transferring the embryogenic tissue culture to culture maintenance medium containing a sufficient amount of nutrients, 0.1 to 5.0 mg/l of auxin, 0.1 to 1.0 mg/l of cytokinin, 10.0 to 40.0 g/l of a sugar selected from the group consisting of glucose, maltose, sucrose, melezitose, and combinations thereof, and a level of gelling agent selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, and 3.5 to 5.0 g/l of AGARGEL, for a sufficient time under suitable environmental conditions to develop a mass of embryogenic tissue having a weight of at least about 100 mg;
• (c) transferring at least about 100 mg of the mass of embryogenic tissue to embryo development medium containing a sufficient amount of nutrients, 5.0 to 33.0 mg/l of abscisic acid, a level of gelling agent selected from the group consisting of 6.0 to 12.0 g/l of agar, 1.75 to 4.00 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, and 3.5 to 6.0 g/l of AGARGEL, and 20.0 to 70.0 g/l of a sugar selected from the group consisting of glucose, maltose, sucrose, and combinations thereof, for a sufficient time under suitable environmental conditions to develop stage 3 somatic embryos;
• (d) separating the stage 3 somatic embryos from the development medium and partially drying the embryos by exposing the embryos to an atmosphere having a high relative humidity for sufficient time to permit the embryos to lose about 25% to 75% of their pre-dried weight;
• (e) transferring the partially dried somatic embryos to germination medium containing a sufficient amount of nutrients, up to 10 g/l of activated carbon, a level of gelling agent selected from the group consisting of 6.0 to 9.0 g/l of agar, 1.75 to 3.50 g/l of gellan gum, 6.0 to 8.0 g/l of agarose, and 3.5 to 5.0 g/l of AGARGEL, and 20.0 to 40.0 g/l of a sugar selected from the group consisting of glucose, maltose, sucrose, and combinations thereof for a sufficient time under suitable environmental conditions to germinate the partially dried embryos;
• (f) converting the germinated embryos into acclimatized plants;
• and
• (g) field planting the acclimatized plants.