Liquid Culture Systems for in vitro Plant Propagation

Mass propagation of conifer trees 391
of Pinus radiata, Pinus pinaster and Picea abies (Timmis, 1998). At
Weyerhaeuser, we have initiated ESM from immature embryos (pre-dome
and dome stages before development of cotyledons) of Douglas-fir
(Pseudotsuga menziesii) and loblolly pine (Pinus taeda). ESM cultures were
initiated onto solid medium with auxin and cytokinin, details of which has
been published earlier. There is no callus stage in ESM induction from
immature embryos. ESMs form directly from heads of early-stage embryos
through cleavage polyembryony. Cleavage polyembryony is natural in
several conifer species, and multiple embryos develop inside the
megagametophyte through this process. ESM cultures are multiplied true-toconifer-type
cleavage polyembryony in the presence of auxin and cytokinin
(Durzan and Gupta, 1987). Induction of ESM has not been achieved in liquid
medium. Semi-solid medium is necessary for ESM initiation, and a
relatively low gelrite concentration (0.1 – 0.2 %) has been found best for the
induction process (Becwar, 1994).
After initiation, ESM cultures are transferred to maintenance medium for
continuation of true-to-conifer-type cleavage polyembryony under lower
concentrations of auxin and cytokinin. At this time osmolality is increased,
from 90-100 to 190-200 mmol kg -1 , with 5 g l -1 myo-inositol and 30 g l -1
maltose (compared with 0.1 g l -1 myo-inositol and 15 g l -1 sucrose in
initiation medium). We have found that the type of sugar is very important
for subsequent embryo development on plates. Early-stage embryos were
able to fully mature only when grown in maltose maintenance medium
(Gupta, 1996). Without increased osmolality treatment, which leads to larger
embryonal heads of early-stage embryos, many genotypes of Douglas-fir and
loblolly pine did not develop good quality cotyledonary embryos (Gupta and
Pullman, 1990).
3. Culture establishment in liquid medium
Liquid cultures offer a number of technical advantages over solid
cultures. Cultures grown in liquid medium have shown a faster rate of
growth. Cultures are bathed in nutrients, which allows rapid uptake of
nutrients by cells and speedy nutrient replacement at the cell surface by
diffusion and movement from outlying liquid. In a gel, this is a slow process,
generating gradients of concentration for each nutrient in the zone of gel
next to the cells, and slowing growth. For these reasons, lower
concentrations of nutrients are usually optimal, compared to those used in
gel media formulation. Due to the faster diffusion rate in liquid systems,
exuded growth inhibitors such as phenolics are rapidly diluted to innocuous
levels. Negative effects on growth are thereby minimized. Liquid media can