Development of in vitro culture and gene transfer techniques in sugarcane (Saccharum species hybrids).
Loading...
Date
1992
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
In vitro cell and tissue culture systems were developed for
sugarcane in order to utilise current transformation techniques to
introduce genes to South African sugarcane varieties, which would
be difficult, if not impossible to achieve in conventional
breeding programmes. Embryogenic calli were initiated in the dark
from stem explants of sugarcane varieties NCo376 and N13, on a MS
medium containing sucrose (20-50 g/l), 2,4-D (2-4 mg/l), casein (1
g/l), inositol (100 mg/l) and agar (9g/l). After 2 months the
somatic embryos were cultured in a light/dark photoperiod for a
further 2 months. The best combination of sucrose and 2,4-D for
callus initiation, and subsequent plant regeneration, was 20 g/l
and 2 mg/l, respectively. Plant yields ranged from 16 to 36
plants per gram fresh weight callus, and the yields were not
significantly increased by the addition of activated charcoal to
the regeneration medium. When plantlets reached a height of 10
cm, they were transferred to autoclaved soil in pots, hardened-off
and placed in the glasshouse.
Suspension cultures were initiated from friable NCo376 calli in
liquid MS medium shaken at 100 rev/min in the dark at 27°C, and
were subcultured every 3-7 days. Protoplasts from various sources
(leaf, calli and suspension cultures) were obtained after
enzymatic digestion in cellulase (20-30 g/l), macerozyme (0,2
g/l), hemicellulase (5 g/l), and sorbitol (0,55 M) in a calcium
and magnesium salt solution. Protoplasts cultured for 48 h
resulted in a loss in viability of 84%.
The potential of the seed as a recipient for direct gene uptake
was investigated, as this eliminated the need for in vitro culture
and plant regeneration. Uptake of [3H] pBR322 DNA by seeds was
demonstrated, and seeds with the testa removed exhibited higher
initial uptake rates than those with intact seed coats. However,
transient expression, using the GUS reporter gene (coding for
bacterial B-glucuronidase) carried on plasmid pBI221, could not be
conclusively shown using the histochemical GUS assay, due to GUS
activity generated by either microbial contamination or endogenous
plant GUS activity. Neither microwaving to eradicate contaminants
nor the addition of methanol (20%) to the GUS incubation buffer
were successful in overcoming positive results observed in control
seeds. An alternative approach to sugarcane transformation, using
PEG-mediated DNA uptake and subsequent transient expression of GUS
by protoplasts was investigated, but microbial contamination was a
persistant problem and no positive results were observed. Further
examination and elimination of endogenous contamination is
required before transformation studies can be continued.
Description
Thesis (M.Sc.)-University of Natal, Durban, 1992.
Keywords
Genetic transformation., Plant cell culture., Sugarcane., Theses--Biological and conservation sciences.