Liquid Culture Systems for in vitro Plant Propagation

392 Pramod K. Gupta & Roger Timmis
be filter sterilized, facilitating aseptic transfer into large closed vessels such
as bioreactors. Handling of plant tissue for harvest and /or transfer is more
amenable to mechanization, which will save labor and time. Scale-up of
liquid cultures also requires less space than for their solid counterparts.
For establishment in liquid culture (suspension culture), 2-3 g ESM are
transferred to 250 ml Erlenmeyer flasks with 20-25 ml of liquid medium.
Flasks are placed on a rotary shaker (90-110 rpm) in darkness at 23 o C. After
7-8 days, old medium is replaced with fresh. Twenty to twenty-five days
after establishment of suspension culture, ESM liquid cultures are poured
into sterile 100 ml measuring cylinders and allowed to settle for 30 minutes.
The supernatant is discarded, and settled ESM cultures measured for
volume. Settled ESM is subcultured in fresh medium at a density of 1:9 (v/v)
by transferring 5 ml settled ESM to a 250 ml Erlenmeyer flask containing 45
ml of fresh liquid medium. ESM suspension cultures are maintained by
regular weekly subculture.
4. Embryo development, maturation and germination
Embryo development is achieved with the combination of activated
charcoal (1.25 mg l -1 ) and ABA (30 mg l -1 ) (Gupta and Pullman, 1990).
Higher concentration of ABA is needed due to adsorption by activated
charcoal. Abscisic acid alone did not inhibit the precocious germination of
cotyledonary embryos. An increased osmolality of the medium is found to
be necessary for late-stage embryo development and maturation. The
osmolality of the late embryo development and maturation medium is
increased to 300-600 mmol kg -1 by adding polyethylene glycol (PEG) MW
4,000-8,000, which was found to be the best osmoticum for good quality
cotyledonary embryo development (Gupta and Pullman, 1991). Several
workers have reported improved conifer embryo development with PEG
(Attree et al, 1994). The quality of cotyledonary embryos was further
improved by a combination of an increased osmolality with ABA (5-50 mg l -1 ),
GA4/7 (5-50 mg l -1 ) and activated charcoal (1.25 g l -1 ) (Pullman and Gupta,
1994). Embryo development and maturation has been achieved using liquid
medium soaked into pads. High osmolality of the medium on the pad has
been maintained by adding a second pad (soaked with higher osmolality
medium) beneath the first. Twenty to fifty cotyledonary embryos were
produced from 1 ml settled ESM suspension culture on development
medium after 5-6 weeks incubation in the dark (Figure 1). The variation in
both quality and number of cotyledonary embryos produced from 1 ml
settled ESM cultures was due to genotypic differences.