Liquid Culture Systems for in vitro Plant Propagation

Mass propagation of conifer trees 395
6. Field performance
Somatic seedlings have now been growing for 9 years on a typical forest
regeneration site in Washington State for clonal demonstration purposes.
Strikingly uniform growth is apparent for somatic seedlings within a clone
(Figure 3) compared with the less uniform zygotic seedlings. No
morphological differences have been observed between somatic and zygotic
seedlings. Recently, we have produced over 100,000 somatic seedlings of
Douglas-fir from a large number of genotypes and established them in soil at
Weyerhaeuser forest regeneration sites for clonal field tests.
7. Culture scale-up for production
Shake flask cultivation is a very effective method of growing
embryogenic cultures for laboratory scale production of clones for fieldtesting.
We have established ESM cultures of Douglas-fir and loblolly pine
in liquid suspension in 1-litre Erlenmeyer flasks. Cultures have been bulked–
up by weekly subculture. Each flask contains over 10,000 early-stage
embryos which may double or triple in number weekly. However, the
occurrence of culture variation from flask to flask, long-term stability of
cultures and lack of control over the culture environment restrict the
usefulness of shake flasks.
For large-scale production of somatic embryos, a bioreactor is one of the
most promising ways for scaling-up the system. Bioreactors offer various
advantages over shake flasks due to the possibilities for automation,
continuous monitoring and control of growth conditions (agitation, pH,
oxygen, and carbon dioxide), larger volume, and maintenance of
homogeneous culture. Many configurations and sizes of vessels have been
used to grow plant cells. Bioreactors for plant cell culture can be classified
according to the type of agitation system used: mechanically agitated
bioreactors, including aeration agitation bioreactors, rotating drum and spinfilter
bioreactors, pneumatically agitated bioreactors, and simple aeration
bioreactors. Non-agitated bioreactors, include gaseous phase (mist)
bioreactors, oxygen permeable membrane bioreactors, overlay aeration
bioreactors and perfusion bioreactors. Aeration-agitation bioreactors are
often referred to as stirred tank and use impellers such as turbines, screws,
paddles and helical ribbons (Ibaraki and Kurata, 2001).
There are several reports of ESM cultures of conifers in bioreactors
(Tautorus et al., 1994, Gupta and Timmis, 1999, Ibaraki and Kurata, 2001).
For different conifer species, different types of bioreactors have been used.
(Table 1). At Weyerhaeuser, several ESM lines of Douglas-fir were grown in