Liquid Culture Systems for in vitro Plant Propagation

Mass propagation of conifer trees 397
There are few reports on cotyledonary embryo development in a
bioreactor using some support for ESM (Table 2). Cotyledonary embryo
development and maturation of White spruce (Picea glauca) has been
achieved in a bioreactor on flat absorbent pads above the surface of a liquid
medium. Medium was continuously supplied to the one end of the pad, while
the spent medium exited by gravity from the opposite end (Attree et al.,
1994). Development medium was pumped from the reservoir into the
bioreactor using a peristaltic pump at the rate of 60 ml per day.
Paques et al. (1995) produced cotyledonary embryo development of
Norway spruce (Picea abies) in a bioreactor using polyurethane layers in
liquid medium. ESM was immobilized in polyurethane layers and placed
vertically in liquid maturation medium. The liquid medium was retained in
polyurethane layers by capillary action, and replaced frequently with fresh
medium. The early-stage embryos in direct contact with liquid medium
(submerged) turned brown while those not in contact (above the surface of
liquid medium) produced cotyledonary embryos.
At Weyerhaeuser, we have also used a bioreactor for cotyledonary
embryo development of Douglas-fir. In the bioreactor, the medium was
supplied semi–continuously from the lower surface of the pads to the
developing embryos on the top. Development medium was pumped from the
reservoir into the bioreactor until it made contact with the lower surface of
pads. Medium was absorbed in the pads by capillary action, and after few
hours, medium was pumped out to the reservoir. This was repeated at regular
intervals until mature cotyledonary embryos developed. Higher yields of
good quality embryos were produced in a bioreactor.
Now it is possible to produce somatic embryos of conifers from liquid
medium in bioreactors. However, different types of bioreactor are currently
required for early-stage embryo growth and cotyledonary embryo
development and maturation. Recently Gorbatenko and Hakman (2001)
produced desiccation-tolerant embryos of Norway spruce in liquid medium
in a shake flask after 8 weeks by subculturing weekly at high density.
8. Embryo selection
The selection of good embryos from among the poorly developed ones
and residual ESM can be a costly bottleneck in production. It is a necessary
step, however, in order to ensure that (1) embryos in the initial mass on a
plate or bioreactor are physically separated for one-by-one processing, and
(2) only those with the highest probability of vigorous germination (“quality
embryos”) move forward in the production process. These two steps are
typically carried out as a single hand motion in which a technician picks out