Induction from some species, particularly pines and Douglas fir, is improved by co-cultivation with the Megagametophyte. 

A haploid nutritive tissue of Gymnosperm  Any class of woody vascular plant which produces naked seeds that are not enclosed within an encapsulated ovary.  seed, of maternal origin, within which the gymnosperm Zygotic Embryos  An embryo derived from the sexual fusion of gametic cells produced by meiosis.  develop.

Induction from pines has generally been easier using immature embryos; whereas Spruces can be induced using mature stored seed. Genotype specificity for embryogenic induction occurs in conifers.

b. Maintaining Embryogenic Cultures.
Embryogenic cultures of conifers are usually maintained on a medium that is similar to the induction medium. Usually, it will include auxin, a cytokinin, as well as a low concentration of a carbon source, and will be maintained on a semi-solidified medium in a Petri dish. These cultures are transferred every 2-4 weeks.

By contrast, liquid cultures are far more effective for rapid Proliferation 

The steps following induction prior to maturation, in which embryogenic cultures divide and grow but do not develop into mature embryo stages. The proliferation step may also be referred to as the maintenance step.

and large-scale propagation. Indeed, these cultures are sometimes transferred as often as once a week. The resulting embryogenic tissues are composed primarily of immature somatic embryos, which greatly resemble immature zygotic embryos. Specifically, they're polarized structures organized into an embryonic region subtended by elongate suspensor cells (see figures). These immature somatic embryos continuously initiate embryos by cleavage polyembryogenesis.

c. Cryopreservation.
A crucial part of the process of Somatic embryogenesis 

A process of initiation and development of somatic embryos in vitro from somatic cells and tissues.

is "cryopreservation", which refers to the storage of germplasm at ultra-low temperatures (i.e. less than -140° C). This process provides for physico-chemical stability of juvenile cultures from a wide genetic array of lines during long-term storage.

Cryopreservation also allows for the reliable recovery of tissue for large-volume production of value-added genotypes. Prior to freezing, Embryogenic cultures 

A plant cell or tissue culture capable of forming somatic embryos and regenerating plants via somatic embryogenesis.

(i.e. maintenance tissue) are incubated with cryoprotecants (i.e. 2 days on an orbital shaker in 0.4 M sorbitol and 5% DMSO) to minimize the formation of intracellular ice and avoid disruption of the cell membrane and wall integrity in the densely cytoplasmic embryogenic head cells. Slow cooling is then applied (i.e. ~0.99° C/min using an isopropanol bath and -80° C freezer) to minimize ice crystal formation via gradual dehydration and adjustment of cell volume. A terminal freezing temperature (i.e. -45° C) is reached at which the cells can be rapidly cooled via immersion in liquid nitrogen (-196° C). Frozen cultures are subsequently stored in the liquid or vapour phase. A rapid thawing process (i.e. 2 minutes at 37-40° C) effects a rapid transition to free water and minimizes the formation of transient ice crystals. Then, the thawed cultures are transferred to a conventional maintenance medium for one day to facilitate removal of excess cryoprotectants. Typically, vigorous regeneration of embryogenic tissue is observed three to seven days after transfer of the cultures to fresh medium.

Each stored vial contains approximately 300 mg of embryogenic tissue. Six to eight vials per Line 

When used in the context of plant propagation, the term refers to a collection of plants produced asexually either from a single plant or part of a plant. They have the exact same genetic make-up.

are placed in long-term storage (primary line banks) at two geographically distinct sites. Secondary line banks contain additional vials for lines selected for research efforts and commercial production.

d. Maturation
In order to encourage the production of mature developmental stages of Conifer 

A cone-bearing vascular plant belonging to the order “Coniferales” which is a sub-class of the botanical group “Gymnospermae”. Conifers used in forestry and reforestation include various species of pines, spruces, cedars, firs, hemlocks and cypress. Conifers produce commercial “softwood”.

somatic embryos, immature somatic embryos must be transferred from an environment that promotes cleavage polyembryogenesis to one containing Abscisic Acid 

A plant growth regulator belonging to the group of stress hormones.  CLICK FOR MORE

(ABA). ABA is a plant hormone which is involved in the maturation of the embryo in the seed. Ideally, this environment would also have a water-stressing component.

This step is usually preceded by treatments containing little or no plant growth regulators. In general, ABA is believed to accumulate during mid-to-late stages of seed development, and will prevent the developing embryo from germinating precociously. Abscisic acid (ABA) levels often decline in the mature dry seed, enabling the Mature embryo 

A mature embryo is one which is capable of germination, given the necessary environmental conditions (temperature, light, water, nutrients, etc.).  CLICK FOR MORE

to germinate upon Imbibition. 

Rehydration of a dry seed or desiccated embryo. This results from the absorption and/or adsorption of water by certain colloids present in seeds or embryos, which result in the swelling of the tissues and activation of enzymatic and physiological processes. Rehydration is usually carried out after a period of storage at low moisture content.

ABA has been found to be essential for inhibiting cleavage polyembryony and promoting the synchronous maturation of many conifer species.

At CellFor, studies of conifer Somatic Embryo 

A plant embryo formed in vitro from vegetative (somatic) cells by mitotic division of cells.  CLICK FOR MORE

development have demonstrated that embryo yields and quality can be substantially improved by progressive increases in ABA concentration and water stress during the course of embryo maturation. Among the potential improvements to embryo quality is enhanced Desiccation 

The drying of a somatic embryo by any means to a Moisture Content  The amount of water present in an embryo.  CLICK FOR MORE less than that of the original hydrated embryo.  CLICK FOR MORE

tolerance and Germination 

A three-phase process starting with uptake of water by a quiescent dry seed, then elongation of the embryo along its axis, then protuberance of a root or shoot radicle through the seed coat.  CLICK FOR MORE

vigor. The accumulation of storage reserves such as lipids and proteins in conifer embryos is controlled by ABA and water stress. Methods for maturation attempt to maximize the deposition of storage reserves, which are essential for vigorous germination. Precise control of variables influencing maturation, including ABA and water stress, can be achieved efficiently using liquid medium supplied to developing embryos in bioreactors. 

e. Maturation Drying
To be commercially viable, Somatic Embryogenesis must be capable of producing millions of embryos throughout the year for delivery to seedling nurseries within a narrow biological-sowing window. However, somatic embryos have a propensity to germinate. This requires that they be stored to prevent early germination. CellFor's proprietary technology allows somatic embryos to be dried and stored, thereby solving this problem.

By storing embryos, it becomes possible to maintain year-round production and stockpiling, which enables delivery of mass quantities of embryos during the very narrow sowing window. Indeed, this short sowing period requires the planting be carried out very quickly, requiring the process to be automated and integrated with nursery sowing technology.

As with most plant species, conifers require seed drying in order to activate the switch from maturation to normal germination mode. Desiccation occurs naturally in zygotic seeds. As a result, somatic embryos show enhanced vigor following desiccation.