|dc.description.abstract||Sugarcane is challenged by a number of phytopathogenic bacteria and viruses that are best
managed by the development of resistant varieties. Genetic engineering is a promising
strategy in such breeding efforts, as it allows novel mechanisms of resistance not available in
any parent germplasm to be introduced into the crop.
DNA sequences encoding cystatin from papaya (Carica papaya), and pleurocidin from the
winter flounder (Pleuronectes americanus) were envisaged as transgenes in this work due to
their theoretical potential to increase sugarcane resistance to viruses and pathogenic or
opportunistic bacteria, respectively. Cystatin is a cysteine proteinase inhibitor. Cysteine
proteinases are used by potyviruses to cleave the polyprotein gene product, an essential step in
the viral life cycle. Constitutive expression of cystatin may therefore lend the host plant
resistance to a range of potyviruses, including the economically important pathogen sugarcane
mosaic virus (SCMV). Pleurocidin is an amphipathic, α-helical, cationic peptide, with broadspectrum anti-bacterial activity at physiological pH. By binding to the cell membranes of both Gram positive and Gram negative bacteria, pleurocidin disrupts the membrane potential,
causing it to become more permeable, especially to cations, leading to death of the bacterial
cell. Initial microbiological bioassays showed that pleurocidin has inhibitory and bactericidal
effects on the organisms which cause leaf scald (Xanthomonas albilineans), gumming disease
(Xanthomonas campestris pv. vasculorum) and post-harvest sucrose conversion in sugarcane,
as well as inhibitory effects against Leifsonia xyli ssp. xyli, which causes ratoon stunting
For transformation vector construction, the cystatin and pleurocidin coding sequences were
altered so that their start codons were in the most favourable consensus context for expression
in monocotyledonous plants. In the case of pleurocidin, an extracellular peroxidase signal
sequence was attached. The prepared sequences were spliced into the vector pUBI510 in
which the gene of interest is driven by the CaMV 35S promoter linked in tandem to a
derivative of the maize ubiquitin promoter. The constructs generated were named pUBI510-cys3
and pUBI510-pleur08 respectively. The plasmid structures were confirmed using
restriction endonuclease analysis and DNA sequencing.
Since the transformation of sugarcane is known to be inefficient, two routes of morphogenesis
for the production of somatic embryos were compared in the transformation procedure. These
were (1) indirect embryo production via callus and (2) the direct and indirect production of
embryos from transverse sections of leaf roll. Field grown sugarcane varieties N12 and
NCo376 were the source of explant material. Plasmids pUBI510-cys3 and pUBI510-pleuro8
were respectively co-delivered by microprojectile bombardment with the antibiotic resistance
selection plasmid pUBIKN containing the neomycin phosphotransferase gene (npt-II).
Cultures were maintained in the dark on selection medium containing various concentrations
of the antibiotic geneticin (G418) for several weeks before being allowed to regenerate in the
light. Plantlets coming through selection were hardened off in the glasshouse when
approximately 100mm high.
Primer pairs for amplification of the cystatin insert were designed in various ways. The primer
pair which ultimately proved most useful was designed to be complementary to the 5' and 3'
ends of the papaya cystatin nucleotide sequence. Primer Premier analysis of a sorghum
cystatin sequence provided additional possible primers. A further pair for potential future use
was devised based on complementarity to conserved regions on maize cystatins 1 and 2,
sorghum, rice, and papaya cystatins. The nucleotide sequence was constructed using the most
common monocotyledon codon permutations for each amino acid. Pleurocidin primers were
designed to be complementary to 5' and 3' regions of the nucleotide sequence encoding the
pleurocidin pre-pro-protein. PCR and RT-PCR protocols for the detection of transgenes and
transcript production in putative transgenic plants were developed using these primers.
No plants survived selection via the callus route, although some were regenerated via direct
embryogenesis. Putative transformed plants were analysed using PCR to test for the presence
of integrated transgenes and Southern hybridization to determine transgene copy number.
Both types of transgene were reproducibly detectable by PCR in DNA from some immature
plants, but results were negative in DNA from those same plants when mature. Southern
hybridization analysis detected the cystatin transgene in DNA from immature plants but no
transgenes were detected in up to 20 µg DNA from mature plants. Single copy constructions
of the transgenes in backgrounds of non-transformed DNA were detectable by both PCR and
Southern hybridization analysis. Overall, PCR, RT-PCR and Southern hybridization results
indicated that the plants regenerated fell into two categories: non-transformed plants that had
survived selection (escapes) and chimaeric individuals with a component of both transformed
and non-transformed cells, in which the transgene had probably become diluted during plant
development under non-selective conditions.
A method for extracting leaf exudates was tested, in conjunction with a cysteine proteinase
assay to detect the presence of cystatin transgenes in the intracellular spaces of sugarcane
leaves of confirmed transformants. Although it could not be applied within the scope of this
project, this assay will prove useful in future work.||en