Liquid Culture Systems for in vitro Plant Propagation

180 M. Berthouly & H. Etienne
We recently observed that increasing the frequency for short immersions
(1 min) stimulated somatic embryo formation and quality in C. arabica.
Thus, average yields of 480, 2,090 and 3,100 embryos were obtained per
1-litre bioreactor, with 60, 79 and 85% torpedo type embryos, for daily
frequencies of 1, 2 and 6 immersions, respectively. Hyperhydricity was not
observed with such immersion conditions. On the other hand, increasing
immersion times by 5 min or more led to a considerable reduction in somatic
embryo production and in their quality, becoming all the more critical as the
immersion frequencies increased. For example, 15 min immersions applied 2
or 6 times per day led to hyperhydrated embryo frequencies of 64 and 90%,
respectively. It is likely that each culture stage requires adaptation of the
immersion length and frequency to obtain optimum results.
4.2 Volume of liquid medium
It is particularly important to optimize the liquid medium volume when
using temporary immersion systems without medium renewal, such as the
twin flasks and RITA � systems or tilting and rocker machines. Lorenzo et al.
(1998) found an optimum volume of medium per explant for sugarcane
shoot proliferation in the BIT � twin flasks system. An increase in
multiplication rate from 8.3 shoots per 30 days to 23.9 shoots per 30 days
was obtained by multiplying the volume of standard medium by ten from 5.0
to 50.0 ml per explant. However, the volume of medium used did not affect
the length of the shoots formed. Higher volumes proved to be less efficient.
According to the authors, the explanation can be found in the secretion of
chemical molecules that stimulate shoot formation, which would seem to be
diluted when large volumes of medium are used. Using the same temporary
immersion system, Escalona et al. (1999) similarly demonstrated with
pineapple that an optimum medium volume exists for shoot proliferation,
which was estimated to be 200�ml per explant for that species. In this case,
larger volumes also led to a drop in the proliferation rate.
4.3 Volume of the culture container
For all temporary immersion systems, the volume of the container, hence
the head space, is much larger than in the containers used for conventional
procedures. Moreover, containers ranging in size from 1 to 20 litres can
usualy be adapted to the system. Krueger et al. (1991) demonstrated that the
large size of their culture container (7 l) had a positive effect on
micropropagation efficiency for serviceberry, notably by avoiding culture
overcrowding and encouraging shoot elongation, when compared to those
obtained in 140 ml baby food jars. Monette (1983) had already shown for