SOMATIC EMBRYOGENESIS TECHNOLOGY
by Steven C. Grossnickle and John Pait

The use of improved seed is an effective way of bringing genetic improvement to forest regeneration programs. Seed orchards are currently producing seeds in large commercial quantities from trees having desired genetic traits. However, improved seed does not provide a method to multiply specific varieties that have desirable traits. Vegetative propagation techniques from full-sib seed provide the best means for doing varietal forestry by multiplying the improved genetic resource developed from tree improvement programs.

Two criteria are considered important for the successful implementation of vegetative propagation systems within an operational forestry program. First, the system must have the ability to preserve superior candidate varieties, so it will require the capacity to maintain varieties in a form capable of regenerating after the minimum period of 5-10 years required to test and select varieties in the field. Second, the propagation system has to be able to multiply selected varieties in large enough numbers at a reasonable cost. If these two criteria can be reasonably met, the selected systems can be implemented.

There have been major advances over the past 50 years in the development of operational vegetative propagation systems for conifer species used in plantations. These systems provide a means of bringing new genetic material into forests through the capture of a greater proportion of the genetic gain inherent within a selected tree species. They also provide a method for multiplying superior varieties and/or families identified in tree improvement programs. Systems utilize one of the following approaches:

Rooted Cuttings - Currently, rooted cuttings are a propagation technique that is available on an operational level to multiply specific varieties that have desirable traits. The primary use of rooted cutting technology is for bulk production of genetically improved materials. This technique is used worldwide to produce tens of millions of rooted cuttings for forest regeneration programs.

Micropropagation through Organogenesis Tissue Culture - Organogenesis is a tissue culture system that relies on the multiplication of shoots or the de novo formation of organs originating from either unorganized callus, preformed shoots, or induced buds. Shoot propagules are placed in an optimal rooting environment and treated in a similar manner as cuttings. This technique has been used in New Zealand forests on radiata pine.

Somatic Embryogenesis - Somatic embryogenesis (SE) is a tissue culture approach where proliferative embryo suspensor masses are established from non-meristematic cells and subsequently cultured to produce organized somatic embryos possessing shoot and root meristems. The term somatic refers to embryos developing asexually from vegetative (or somatic) tissue. This method has been used in horticulture and agriculture on a limited basis, and is now being used to a greater scale in forestry. 
SE is the only vegetative propagation technology that provides long-term preservation of the selected genetic component of a conifer species that can be used for extended timeframes within an operational forestry program.

Basic Laboratory Protocols for SE
In general, the SE process is divided into several laboratory steps, which are performed under sterile conditions to prevent microbial contamination.
Culture initiation - mature zygotic embryos are dissected from the seed and placed onto semi-solid medium containing plant growth regulators.

Proliferation - maintenance of embryonal suspensor mass, which is characterized by the presence of early-stage somatic embryo structures that are analogous to those occurring during normal seed development. This is followed by a multiplication step when the tissue multiplies and develops as early-stage somatic embryos. Embryogenic cultures can be proliferated in a juvenile form for long periods of time to produce unlimited numbers of propagules from the same variety. At this point tissue can be allowed to continue to grow or it can be placed into long-term storage.

Cryopreservation - a means whereby germplasm can be stored. The embryogenic tissue is treated with cryoprotectants, frozen to -35oC under a controlled freezing rate, and then subsequently stored in liquid nitrogen (-196oC) (Figure 1). Cryopreserved tissue can be stored indefinitely and then regenerated within a few weeks after a simple thawing process. This long-term storage option offers a distinct advantage of somatic embryogenesis tissue culture over rooted cuttings and organogenesis tissue culture.

Maturation - advances the development of somatic embryos by exposing tissue to phyto hormones and controlled environmental conditions. Within a period of a few months, they are transformed into mature somatic embryos that are analogous to zygotic embryos.

In vitro germination - final lab step in which embryos are placed on germination media under controlled environmental conditions. In vitro germination occurs within a week and proceeds to the development of true needles. At this point young somatic seedlings can be transferred to ex vitro nursery conditions.

Nursery and Field Performance
From the early 1990s until the present, germinants from SE technology have shown continued improvement in their development into high-quality somatic seedlings in the nursery. Somatic seedling propagation technology has also been successfully integrated into both the bareroot and container seedling production systems. The initial response of germinants to the nursery environment will have a profound influence on subsequent morphological development. Recent nursery performance of somatic seedlings has shown that a proper nursery cultural environment (nutrients, temperature, and moisture) during the initial establishment stage will result in normal morphological development of seedlings. Reforestation site trials have found that somatic and zygotic seedlings have comparable field performance as a stocktype.

Integration into Tree Improvement Programs
SE technology provides an opportunity to capture value-added traits at the individual tree or family level. Testing of progeny from selected parents will capture additional gain for improved performance such as growth and yield, wood quality, plus stress, pest, and disease resistance. Thousands of varieties can be produced for field trials from selected families having desirable genetic traits. Embryogenic cultures from these varieties can be cryopreserved for long-term storage until field selections are made. Ultimately, a population of varieties, large enough to ensure genetic diversity, can be selected based on field performance criteria. From this type of selection program, seedling suppliers can offer elite varieties of loblolly pine seedlings with yield improvement averaging 42% (Figure 2). The selected varieties are removed from cryostorage and produced as somatic seedlings in the tens of millions, and are then deployed operationally to reforestation sites as diverse genetic mixtures. 

Operational SE Production System
Commercial acceptance of a novel technology such as SE requires the ability to develop and implement a successful operational use for the technology. The following key components must be addressed during this stage:
• Development of a cost-effective manufacturing process
• Delivery of high-quality products that provide predictable and reproducible performance
• Technology validation and promotion in the marketplace
During the past decade, significant progress has been made towards developing reliable, high-volume, cost-effective SE production systems. Organizations are working on commercialization programs for spruce species, Douglas fir, loblolly pine and radiata pine. 

The Future of SE Technology
Forestry companies, advance seed production companies, and government organizations around the world are currently working on bringing tissue culture technology to a point where it can produce conifer somatic seedlings, on a cost-effective basis, with desired genetic characteristics. In the southeastern US the returns on planting elite varieties of loblolly pine seedlings produced from tissue culture technology are evident in more tons per acre grown per year, with fewer diseased stems, higher quality, and straighter logs with small knots, which will command the highest prices in the market. A southeastern US landowner can expect to realize a 10-18% return on investment in seedlings produced from these elite loblolly pine varieties, and harvest revenues that may be 75% greater in terms of 2006 dollars (net present value) than revenues from traditional orchard stock. 

In the North American forestry market, CellFor Inc. and Arborgen are the two companies currently using SE technology to produce conifer seedlings for the commercial marketplace. For the 2008 planting season, CellFor Inc. will produce 10 million seedlings, while Arborgen will produce 500,000 to 1 million seedlings of southern yellow pine. In the southern US, the current market for southern yellow pine seedlings is 1.2 billion seedlings on an annual basis. It is projected that the marketshare of elite varieties of yellow pine will be 5% within the next 2-3 years.

With a supply chain and potential economic returns, SE is now a viable system for producing elite varieties of conifer species for the forestry industry.

Steve Grossnickle is a Senior Manager at Cellfor leading an effort to integrate somatic embryogenesis propagation technology for conifer species into nursery operations. Steve can be reached at 250-544-0492 ext. 223 or sgrossnickle@cellcor.com.