Abstract
The microprojectile bombardment of immature embryos has proven to be
effective in transforming many indica rice varieties. One of the drawbacks
of using immature embryos is the requirement of a large number of high quality
immature embryos, which itself is a tedious and laborious process. To circumvent
these problems, we have developed a procedure, using indica variety TN1
as a model that generates highly homogenous populations of embryogenic subcultured
calli by selectively propagating a small number of regeneration-proficient
calli derived from seeds. Thousands of embryogenic calli were produced from
50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were
regenerated per 500 embryogenic calli bombarded. The convenience and reliability
offered by this transformation system has made transformation of indica
rice a routine procedure.
Abstract
The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv.
oryzae race 6, was isolated by positional cloning. Fifty transgenic rice
plants carrying the cloned Xa21 gene display high levels of resistance to
the pathogen. The sequence of the predicted protein, which carries both
a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests
a role in cell surface recognition of a pathogen ligand and subsequent activation
of an intracellular defense response. Characterization of Xa21 should facilitate
understanding of plant disease resistance and lead to engineered resistance
in rice.
Abstract
The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which
infects cassava plants (Manihot esculenta) and has been characterized as
a plant pararetrovirus belonging to the caulimovirus subgroup [9] . Two
DNA fragments were isolated from the viral genome and fused to the uidA
reporter gene to test promoter activity and to map the transcription start
site. Both fragments were able to cause high level of gene expression in
protoplasts isolated from cassava and tobacco cell suspensions. The pattern
of expression of the CVMV promoters was also analyzed in transgenic tobacco
and rice plants, and revealed that the GUS staining pattern was similar
in both plants. The CVMV promoters were active in all plant organs tested
and in a variety of cell types suggesting a near constitutive pattern of
expression. In both tobacco and rice plants GUS activity was highest in
vascular elements, in leaf mesophyl cells, and in root tips.
Abstract
A protocol was established for the introduction of DNA into embryogenic
suspension-derived tissues of cassava via microparticle bombardment, for
the selection of genetically transformed cells, and for the regeneration
of fully transgenic plants from these cells. The plasmid DNA used for bombardment
contained a gene encoding neomycin phosphotransferase (nptII) and a gene
encoding ß-glucuronidase (uidA). Selection of bombarded tissue with
paromomycin resulted in the establishment of putative transgenic embryogenic
calli. In most of these calli ß-glucuronidase (GUS) was detected histochemically.
Molecular analysis of paromomycin-resistant embryogenic calli and of plants
regenerated thereof confirmed the stable integration of bombarded DNA into
the cassava genome.
Abstract
Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties
IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3
were established within 6-8 weeks from 3-4 week old calli derived from mature
seeds. Transgenic rice plants were obtained by introducing a plasmid carrying
genes encoding hygromycin phosphotransferase (hph, conferring resistance
to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV
35S promoter, via particle bombardment of embryogenic suspensions. The effect
of osmotic conditioning on transformation was evaluated. Regenerated plants
were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth
of mother plants (R0) was normal and seeds were produced. Southern blot
analysis of R0 and R1 plants showed that hygromycin resistant plants contained
intact hph genes that were inherited in a Mendelian fashion. A protocol
for a simple, efficient, repeatable, genotype- and environment-independent
Indica rice transformation system is described.
Abstract
The genomes and ORFs of 36 geminiviruses were compared to obtain phylogenetic
trees and frequency distributions of all possible pairwise comparisons with
an objective to classify geminiviruses. Such comparisons show that geminiviruses
form two distinct clusters of leafhopper-transmitted viruses that infect
monocots (subgroup I) and whitefly-transmitted viruses that infect dicots
(subgroup III), irrespective of the part of the genome considered. Of the
two leafhopper-transmitted viruses that infect dicots, the tobacco yellow
dwarf virus has a sequence most similar to subgroup I viruses, and that
of beet curly top virus differed depending upon the ORF considered. The
distributions of identities within subgroups are significantly different
suggesting that the taxonomic status of a particular isolate within a subgroup
can be quantified. All the recognized strains of any one virus have greater
than 90% sequence identity. It was observed that the 200 nucleotide intercistronic
regions of geminiviruses are more variable than the remainder of the genome.
The amino acid sequences of the coat protein (CP) of subgroup III viruses
are more conserved than the remainder of the genome. However, a short N-terminal
region (60-70 amino acids) of the CP is more variable than the rest of the
CP sequence and is a close representation of the genome. PCR primers based
on conserved sequences can be used to clone and sequence the N-terminal
sequences of the CP of the geminiviruses, and is sufficient to classify
a virus isolate. A possible taxonomic structure for geminviruses is proposed
after considering the sequence comparisons and biological properties.