Biotechnology, Breeding and Seed Systems for African Crops
Biotechnology
Development of Agro-bacterium-mediated clean gene
(marker free) technology for rice transformation using a
novel dual binary plasmid pgreen / psoup
A.S.
Afolabi, J. Snape & P. Vain
The possibility of
out-segregating marker genes (during sexual crossing) to produce
transgenic rice plant using Agrobacterium
was investigated and demonstrated to be feasible using a novel pGreen/pSoup
dual binary system. We cloned into one T-DNA (pSoup plasmid) expression
units containing the aph1V and
gfp genes and into a second
T-DNA (pGreen) the expression units containing the bar and the gus genes.
Both plasmids were introduced into Agrobacterium
strain AGL1 which was used to transform
calli derived from matured embryo of Nipponbarre rice seeds.
Transformed calli were selected using Hygromycin and/or Phosphinotricin
(PPT) and hundreds of independently transformed fertile plants were
regenerated. Transformation efficiency with Agrobacterium
strain AGL1, when both plasmids were selected for, was found to range
from 23-44%. Co-transformation efficiency was
100% when both plasmids were selected for with Hygromycin and L-
Phosphinotricin (PTT) and 71% when only pSoup was selected using
Hygromycin selection. Fertility in all cases was more than 85%. Phenotypic
and genotypic analyses were perfomed on progenies using 64 T1
seeds from 68 independent T0 plants (50 plants selected on
both Hygromycin and PPT and 9 plants selected on Hygromycin alone and 9
plants selected on PPT alone). Plants were phenotypically observed for
green fluorescence (GFP) and GUS coloration using UV microscopy and GUS
histochemical assay, respectively. Four phenotypic categories were
obtained: (1) GFP+ and GUS+ progenies that contained and expressed both
T-DNAs from the pGreen and pSoup plasmids; (2)
GFP+ and GUS- progenies
containing and expressing the T-DNA from pSoup plasmid and not
expressing or not containing the T-DNA from the pGreen plasmid;
(3) GFP- and GUS+ progenies containing
and expressing T-DNA from pGreen
plasmid and not expressing or not containing T-DNA from pSoup plasmid;
(4) GFP- and GUS- not containing or not expressing the T-DNAs in plasmids pGgreen
and pSoup. Polymerase chain
reaction (PCR) and Dot blot analyses were used
to determine the presence of the four transgenes (bar,
gusA, gfp and aph1V) in all progenies exhibiting no phenotype. Both
the PCR and dot blot data showed that unlinked T-DNA integration could
be obtained as well as linked integration. Of the 50 To
plants (PPT & Hygromycin selection) evaluated at the structural
level, 56% showed unlinked T-DNAs. The observed genotypic segregation
analysis data were subjected to
statistical analysis and compared to the theoretical Mendelian values. Our
results indicated that Agrobacterium-
mediated “clean gene” technology using pGreen / pSoup dual binary
vector can produce marker free-transgenic rice plants. Most of the
observed and analysed integration patterns of the T-DNAs in different
combination (up to 4 loci and 30 different permutations) falls within
two and three loci Multiple Cry
genes for avoiding build-up of resistance among insects to Bt
toxins: Learning from Bt-rice I. Altosaar & M.
Zaidi. To promote the sustainable use of Bt
crops, Bt cultivars should only be released if they contain two Bt
toxins, both expressed at a high dose in the transgenic plant. If
insects that are able to survive on a plant with one high-dose toxin are
rare, then insects that are able to survive on plants with two high-dose
toxins will be even rarer. If such insects must be homozygous for
resistance alleles for two different genes, and if the frequency of the
allele for resistance of each gene is 10-3, then insects of
the genotype R1R1R2R2 will
occur at a frequency of only 10-12, i.e., 1 out of 1
trillion. Because such insects will be very rare, fewer susceptible
insects will be needed to ensure that resistant insects do not mate with
each other. Fewer refuge fields may then be necessary. Our lab has
synthesized two plant codon optimized coding sequences for the high-dose
expression of Cry1Ab and Cry1Ac. These have been deployed in one-toxin
Bt-rice field trials with success for the time being but more synthetic
sequences are needed to test if pyramiding Cry genes can prevent
resistance from occurring. We are adding three new Cry genes to our
research repertoire, two 1C sequences varying in G-C content (45%, 65%)
and a plant-optimized 2A coding sequence (61%). For example, the
re-synthesis of Cry1C coding sequence, on assembly still contained 16
basepair errors. The clone (pTY185.1) containing the 1.9 kb coding
sequence was cut into two halves, each half subcloned separately into a
cloning vector (pPCR script and pGEM4Z for their convenient restriction
sites). The repair of
mistakes was done by site directed mutagenesis using the “Quick Change
Mutagenesis™” kit from Stratagene. Primer pairs were complementary oligonucleotide sequences
between 25 and 40 bases. They
were designed such that 10-15 bases of correct sequence flanked the
desired mutation site on either side. During the course of this repair
three new errors appeared. These
were the errors incorporated by Pfu Turbo DNA Polymerase used in the
repair process. Luckily, swapping them with the original clone,
pTY185.1, could repair these new errors.
Today, all errors have been removed and their verification
through sequence analysis is in progress. The two halves of the coding
sequence will be assembled together and cloned into an expression
cassette for E. coli (Codon
plusä from Stratagene) and the bioactivity of the
expressed protein will be tested against target podborers and stemborers.
Plant transformation will target these coding sequences to cowpea,
sorghum and tropical maize. Marker
assisted selection (MAS) deployment of the novel CMD resistance gene (CMD2) in African cassava gene pools O. Ariyo,
A.G.O.
Dixon, N. Morante, H. Ceballos & M. Fregene The
Rockefeller foundation funded project on the molecular mapping of genes
conferring resistance to the molecular markers tightly associated to a
new source of resistance cu in cassavas designated CMD2 have been identified. The
effectiveness of CMD2 against
a wide spectrum of virus strains in sub Saharan Africa, including the
aggressive Ugandan variant (UgV), makes its deployment very appealing in
protecting cassava production against the ravages of CMD in Africa.
Deploying CMD2 widely in African cassava gene pools requires breeding for
other biotic stresses, particularly CBB and CGM, as the CMD2 donor
parents have been found to be very susceptible to both.
A marker-assisted selection (MAS) project was initiated to
combine CMD2 with good genes for CBB and CGM resistance. MAS drastically reduces, by as much as 95% (for four genes),
the number of progenies carrying all required genes at the seedling
stage thereby increasing the efficiency and cost-effectiveness of
breeding. Several good
sources of CBB resistance and an excellent source of CGM resistance were
crossed to the donor genotypes TME3 and TME9, two sources of the CMD2
gene, or their resistant progenies. The progenies were evaluated by MAS
and phenotypically for CMD, CBB, and CGM.
The cost of using markers vs. conventional, phenotypic selection
against no markers was calculated to determine the cost effectiveness of
MAS. The logistics
and practicality of setting up and running a MAS laboratory is also
discussed. R. Buruchara,
G. Mahuku, S. Mayanja, J. Mukalazi & A. Levesque Pythium root rot caused by a number of Pythium
spp is the most destructive soilborne disease of beans (Phaseolus
vulgaris L) in East and Central Africa and can result in complete
crop failures in some seasons. Identification of species belonging to
the Pythium genus is both slow
and difficult using morphological or pathogenic characteristics. Rapid
and accurate detection assays are required because correct species
identification is important as a basis for identification and
development of host resistance and other management strategies. The
objective of this study is to adapt a relatively fast and accurate
diagnostic method known as “Reverse Dot Blot Hybridization” (RDBH)
for species detection and identification. RDBH is based on
species-specific oligonucleotides designed and blotted onto a membrane
array which currently only includes species occurring in temperate
regions. The first step for the application of the RDBH in tropical
regions of Africa requires validation, through extensive
characterization, so that Pythium species prevalent in Africa are represented on the membrane
array. DNA from over 400 Pythium
isolates, obtained from soil and diseased bean plant samples in Uganda,
Kenya and Rwanda, were amplified with Pythium
group–specific primers that target the internally transcribed spacer
(ITS) region of the ribosomal genes. Direct sequencing of the PCR
fragments was done for over 150 isolates using primers that annealed
inside of the first fragment. The sequences were analyzed by comparing
them to sequences of known Pythium
species from a Pythium
database managed by Dr A. Levesque. Sequence analysis of the isolates
identified 12 different species with P.
ultimum Var. ultimum being
the most prevalent. Other species identified included P.
irregulare, P. dissotocum, P. indigoferae, P.
oligandrum, P. salpingophorum, P. spinosum, P. torulosum P. vexans, P.
nodusum, P. deliense/aphanidatum, and P. pachycuale.
Interestingly, P. oligandrum,
a known biocontrol agent, was among the Pythium species isolated. Four
new putative species found in association with beans were among the neo
(type) strains, implying they could be novel. A number of
isolates were identified as Mortierella
spp, a common saprophyte that can be isolated on Pythium-specific
media and which is culturally and morphologically similar to Pythium
spp. Implications for resistance breeding imposed by species diversity
and distribution are discussed. Towards a
maize-based edible vaccine: LT-B-producing maize protects mice against Escherichia
coli heat labile enterotoxin (LT) and cholera toxin (CT) R. Chikwamba, J.
Cunnick, D. Hathaway, J.
McMurray, H. Mason & K. Wang Recent
advances in plant transformation have allowed the use of crop plants
beyond their traditional uses of feed and fiber.
We produced a functional heat labile enterotoxin (LT- B) subunit
of Escherichia coli in maize.
LT-B is a multimeric protein that presents an ideal model for an
edible vaccine, displaying stability in the gut and inducing mucosal and
systemic immune responses. Transgenic maize was engineered to synthesize the LT-B
polypeptides, which assembled into oligomeric structures with affinity
for GM1 gangliosides. We
orally immunized mice by feeding transgenic maize meal expressing LT-B
or nontransgenic maize meal spiked with bacterial LT-B.
Both treatments stimulated elevated IgA and IgG antibodies
against LT-B and the closely related cholera toxin B subunit (CT-B) in
serum, and elevated IgA in fecal pellets.
The transgenic maize induced a higher anti-LT-B and anti-CT-B
mucosal and serum IgA response compared to the equivalent amount of
bacterial LT-B spiked into maize. Following
challenge by oral administration of the diarrhea-inducing toxins LT and
CT, transgenic maize-fed mice displayed reduced fluid accumulation in
the gut compared to non-immunized mice.
Moreover, the gut-to-carcass ratio of immunized mice was not
significantly different from the PBS (non-toxin) challenged control
group. We concluded that
maize-synthesized LT-B had features of the native bacterial LT-B such as
molecular weight, GM1 binding ability, and induction of serum
and mucosal immunity. We
have demonstrated that maize, a major food and feed ingredient, can be
efficiently transformed to produce, accumulate and store a fully
assembled and functional candidate vaccine antigen. The
genetics of virulence of the maize streak mastrevirus
(MSV) R. Edema, D.T. Gordon & D.M. Bisaro Maize Streak Mastrevirus (MSV) isolates differ in the severity of
symptoms they incite on maize. Virulent forms of the virus incite severe
stunting and chlorosis on infected plants and drastically reduce yield. Of major importance to breeding programs
is the knowledge of whether current resistance is durable when
challenged with the most virulent MSV isolates. However, the relative
virulences of MSV isolates across the geographic range of MSV is
unclear. A reliable method for identifying MSV virulent types would,
therefore, be of considerable value. We focused on developing a means of
identifying virulent MSV isolates based upon genetic variation. The genomes of four isolates were cloned and sequenced. Dimeric cloned
DNA of each isolate was infective when inoculated to susceptible maize
by vascular puncture inoculation. Clone symptom severity was classified
as mild (MSV-KL), moderately mild (MSV-Z), moderate (MSV-KH, or severe (MSV-Km).
Virulence determinants were mapped using
chimeric clones constructed from parental clones pMSV-KL (mild) and pMSV-Km
(severe) employing restriction endonuclease fragments. Complementary
exchange of restriction fragments between the genomes of pMSV-Km
(severe) and pMSV-KL (mild) demonstrated that determinants of virulence
were present in several regions of the genome, including those encoding
the coat protein, replication associated and replication initiator
proteins and the non-coding long and short intergenic regions. Analyses
of these sequences, along with MSV sequences from the GenBank for
isolates with reported symptom phenotypes, were conducted for motifs
associated with virulence. The analysis involved various on-line tools
and databases. Phylogenetic analyses of the amino acid sequences of the
replication-associated (Rep and RepA), movement and coat proteins failed
to reveal distinct African regional strains or symptom phenotype motifs.
It was concluded that MSV isolates from Africa exist as a common,
multi-allelic genome population without a simple genetics of virulence. Genetic mapping
of a dominant gene conferring resistance to the cassava mosaic disease (CMD) M.
Fregene, A.O. Akano, A.G.O. Dixon, C. Mba & E. Barrera
The
cassava mosaic disease (CMD) is the most important disease of cassava in
Africa, and a potential threat to the crop in Latin America where the
disease is still not known but the vector has recently been found. Host
plant resistance is the principal method of control, and was first
identified in 3rd back cross derivatives of an inter-specific
cross between cassava and M. glaziovii.
It is thought to be polygenic with a recessive component.
Recently, several Nigerian cassava landraces have been
identified that show very high resistance to CMD.
Classical genetic analysis and molecular genetic mapping revealed
a single dominant gene control of resistance.
A bulk segregant analysis (BSA) approach was used to identify two
simple sequence repeat (SSR) markers linked to the CMD resistance gene.
The SSR marker is located on linkage group R of the male
parent-derived molecular genetic map. The gene has been designated CMD2
and is flanked by one SSR marker and a RFLP marker at 2 and 9 cM
respectively. The second
SSR marker shows no recombination with the CMD2.
The use of markers linked to CMD2
for marker-assisted breeding of CMD resistance to increase the
efficiency of breeding in Africa and Latin America is discussed. Efforts
are also ongoing to clone CMD2 and genes expressed downstream that are the molecular basis of
resistance using positional cloning and the serial analysis of gene
expression. Engineering insect resistance into maize using Bacillus thuringiensis (Bt) cryiac gene fused to the c-terminal
(galactose-binding) of the ricin B-chain D.
Gahakwa, L. Mehlo, A. Gatehouse, J.P. Du, N.T. Loc, J. Gatehous & P.
Christou The bacterium Bacillus
thuringiensis (Bt) is the source of d-endotoxins
(or Cry proteins) with potent insecticidal activities. These proteins
have been widely used for crop protection, both through topical
application and the expression of Bt genes in transgenic plants. To
avoid the evolution of insects showing resistance to Bt toxins, much
effort has been directed towards engineering crystal proteins with novel
activities. The binding property of CryIAc was modified by adding the
galactose-binding domain of the non-toxic ricin B-chain. Transgenic
maize plants expressing the CryIAc-ricin fusion protein were generated
by the bombardment of embryogenic calli with fusion constructs. Southern
and western blot analyses showed that the transgene was stably
integrated and expressed into the maize genome and was transmitted to
progeny over at least three generations. The efficacy of the transgenic
maize plants was tested using insect bioassays. The fusion protein was
found to be toxic to Chilo
supprelissalis (rice stem borer), Spodoptera
littoralis (cotton leaf worm, which has been reported to show
tolerance to some Cry1 toxins), Cicadulina
mbila (leafhoppers) and
Rhopalosiphum padi (bird
cherry oat aphid).
The last two represent
homopteran insects that are not normally affected by the CryIAc toxin.
These results show that the activity of the CryIAc toxin was enhanced
and the range of target insects was broadened.
Molecular, expression and bioassay data will be discussed in
the light of our findings. Bioassays to
characterize and dissect mechanisms of resistance to striga C. Grenier,
A. Mohammed, P.J. Rich, T. Housley & G. Ejeta We have developed three in vitro techniques that reveal critical stages in host-parasite
interaction during infection of host plants by Striga (Striga ssp.). We routinely
employ these bioassays to evaluate host plants for resistance to Striga
and to characterize the specific mechanisms involved in the defense
reaction of host genotypes. The agar gel assay (AGA) and the extended
agar gel assay (EAGA) involve placing conditioned Striga seeds in an
agar layer and measuring Striga germination and haustorial production in
response to stimuli exuded by host genotypes. The paper roll assay (PRA)
involves growing sorghum seedlings with their roots between rolled
layers of germination paper and allows observation beyond germination
and haustorial development associated with early attachment and
penetration. Host responses to Striga parasitism using these assays
reveal the potential existence of at least the following four separate
mechanisms of Striga resistance: 1) low production of Striga seed
germination stimulants and evidence of germination inhibitors; 2) low
production of the signal required for haustoria initiation; 3) a
hypersensitive response reaction characterized by a distinct necrotic
area on the host root at the attachment site that discourages parasitic
establishment; and, 4) an incompatibility response where parasite
development is arrested with no apparent necrosis on the host root. In
addition to breaking down resistance mechanisms, these assays offer
several additional advantages to a plant breeding program including: a)
rapid and cost effective screening of host germplasm to identify useful
genetic variants; b) cataloguing host of germplasm on the basis of gene
sources for different mechanisms of resistance; c) introgressing and
pyramiding multiple resistance genes into a desired cultivar; d) serve
in mapping of genomic regions associated with specific resistance
mechanisms for marker assisted introgression; and, e) targeting
isolation and cloning of different Striga resistance genes. Generating
striga resistant maize:
Identification of sources of resistance in wild relatives A.L.
Gurney, D. Grimanelli, S.J. Hearne, F. Kanampiu,
D. Hoisington, J.D. Scholes & M.C. Press Parasitic weeds of the genus
Striga can devastate grain
production in small-holder farms in Africa. Complete resistance to Striga
infection has not been identified for cereals, moreover, genetic
variation in maize for Striga resistance/tolerance is extremely limited. There is an urgent
need to understand the physiological and molecular basis of the cereal-Striga
association to help provide strategies for the long-term control of Striga.
One approach to generate Striga-resistant
material is to identify sources of resistance from wild relatives of
maize. The genetic potential of a number of diploid and tetraploid Tripsacum
accessions was assessed to determine their value as a source of alleles
for maize breeding. Novel sources of resistance were observed in
tetraploid Tripsacum dactyloides.
Tripsacum did not support Striga
growth as development of the parasite was arrested soon after attachment
and initial development of the haustorium. The haustorium is a unique
organ that provides a physiological bridge between host and parasite,
acting as a conduit for host derived water and solutes. Vascular
continuity was established between parasite and host but histological
analysis demonstrated inferior haustorial development on Tripsacum
compared with maize. The hyaline body, a metabolically active region of
cells within the haustorium, was either absent or poorly developed.
These results strongly suggest that Striga
fails to develop on Tripsacum
due to nutrient starvation. Recent results indicate that Tripsacum
may produce a negative signal that impairs normal haustorial development
and function. Evaluation of
a number of maize-Tripsacum
hybrids in the laboratory indicated that resistance to Striga
decreased as the proportion of maize present in the genome increased.
The use of maize-Tripsacum addition lines may allow putative locations for resistance
gene(s) to be identified. This approach will help to assess the
feasibility of introducing Tripsacum-based
resistance into maize using conventional wide cross techniques.
Attention is also focused on identifying the resistant trait in Tripsacum. Chemistry of Desmodium
root exudate and Striga control: research status and prospects A. Hassanali, M.K. Tsanuo,
A.M. Hooper, Z. Khan, F. Kaberia, J. Pickett & L. Wadhams Two
types of isoflavanoid constituents have been found in compositionally
complex root exudates of Desmodium
uncinatum: germination stimulants and haustorium inhibitors.
The combined action of these allelochemics provide a novel, in
situ mechanism of continuously depleting Striga
seeds from the soil, so far demonstrated in experimental Desmodium-maize
intercrops. Some of the
allelochemics have been isolated and characterized.
The differences in activities of these compounds suggest
relatively specific structural requirements for germination stimulatory
and haustorium inhibitory activities.
The implications for screening for Desmodium-type Striga
suppression attribute and for a biotechnological approach to
transferring this attribute to other crops will be highlighted.
Specifically, the presentation will address the following topics:
1) the diversity of Desmodium root chemistry and role, if any, in Striga suppression; 2) the occurrence of isoflavanoid biochemistry
in Leguminosae and 3) other families and probability of finding
Desmodium-type secondary compounds; and prospects of transferring
Desmodium biochemical attributes to other crops. Striga free maize: Introducing Tripsacum-based
resistance via an integrated approach S.J.
Hearne, A.L. Gurney J.P. Brizard, C. Brugidou, F.
Kanampiu, P.O. Mbogo, M.C. Press, J.D. Scholes & D. Hoisington Tetraploid Tripsacum
dactyloides (L.) exhibits
resistance and tolerance to Striga
under field and laboratory conditions.
The development of Striga is
impaired soon after attachment. This
perturbation of Striga
development has been observed in crosses between maize and tetraploid Tripsacum indicating that the trait may be transmittable.
To assess the potential for trait transmission via wide crosses,
a large number of maize-Tripsacum hybrids with differing chromosome numbers were screened in
glasshouse experiments in western Kenya.
Those plants with 15 or more Tripsacum chromosomes performed very
well under conditions of Striga infestation. A range of maize-Tripsacum addition lines are being screened in the laboratory in
order to determine whether the trait can be localised to a chromosome or
group of chromosomes of Tripsacum.
This data will help ascertain how complex the trait is and help
determine whether transmission of the trait from Tripsacum
to maize is possible via wide crossing.
In the absence of wide crossing, introduction of the trait into
maize may be possible but first we must identify those characters
responsible for the interesting phenotype.
Thus, we are using a combined approach incorporating both
proteomics and genomics in order to elucidate the basis of resistance. Will
transformation systems for the large seeded grain legumes, peas, lupins
or chickpeas be adaptable to cowpea? T.J.V. Higgins,
L Molvig, W. Tate, S. Gollasch & L.M. Tabe It
is now possible to transform many large-seeded grain legumes. Although
the rate of recovery of transgenic lines together with transmission to
the next generation is low, the systems are robust enough to allow
transfer of the technology from lab to lab. Low efficiency, means that
0.3 to 1% of the starting explants will result in a unique
transformation event. Thus,
it is still a very laborious process and it is clear that a large-scale
effort is needed for any pulse legume project.
We find that it is important to avoid a callus phase during
regeneration and transformation. Other
features that are critical to success are selection of an explant that
regenerates in tissue culture with a high frequency.
The choice of selection agent, medium type and hormonal regime,
while important, are of secondary significance.
Three approaches to transformation of legumes can be illustrated
with peas and chickpeas expressing a bean a-amylase
inhibitor gene and lupins expressing a gene for a rumen-stable, high
sulfur, amino acid-containing protein.
The aims of this research are to improve field performance and
seed quality of peas and chickpeas.
Enhanced seed protein quality of lupins is aimed at more
efficient ruminant animal production.
These approaches to pulse transformation are being adapted to
cowpea. Control of the
witchweed Striga hermonthica
by intercropping with Desmodium spp. Z.R. Khan,
A. Hassanali, W. Overholt, T.M. Khamis,
J.A. Pickett & L.J. Wadhams It was discovered through field work in Kenya that
inhibition of witchweed, Striga
hermonthica, a in maize-Desmodium
uncinatum intercrop was significantly greater than that observed
with other legumes, e.g. sun hemp, soybean and cowpea. The objective of
this study was, therefore, to investigate the mechanisms by which D. uncinatum inhibits the parasitic plant.
This could be by increasing available nitrogen, by offering shade
or by an allelopathic effect, all of which are known to give some
control of these parasitic weeds. A field trial was established to
determine the role of shading, nitrogen and allelopathy in Striga
suppression by D. uncinatum.
The treatments were maize intercropped with D.
uncinatum without/with 120 kg nitrogen per ha, maize monocrop
without/with 120 kg nitrogen per ha, and maize monocrop with artificial
ground shading made of maize straw with and without 120 kg nitrogen per
ha. Emergence and attachment of Striga
to maize was measured and maize plant height and grain yield were
recorded. To investigate the possibility of an allelochemical mechanism,
effluent from D. uncinatum roots was used to irrigate maize planted with
approximately 3,000 Striga seeds.
D. uncinatum and maize were
planted in different sets of pots in previously autoclaved soil.
Distilled water dripped into D.
uncinatum containing pots which were placed above the pots
containing maize. Comparison was made between maize plants irrigated by
root exudates of D. uncinatum
against those irrigated with water passing through pots with autoclaved
soil but without D. uncinatum.
Emergence and attachment of Striga
to maize was monitored in all treatments. In field trials, although soil
shading and addition of nitrogen fertiliser showed some benefits against
Striga infestation, a clear
allelopathic mechanism was also observed.
This was confirmed by a dramatic reduction in Striga
infestation when eluate from D.
uncinatum roots was introduced into pots of maize growing in soil
seeded with high levels of Striga.
Growth of the parasitic weed was almost completely
suppressed, whereas extensive infestation occurred with control eluate.
The allelopathic mechanism was found to involve inhibition of
development of haustoria of Striga. Work is ongoing to identify the compounds, released from the
D. uncinatum roots, involved
in suppression of the parasite. The sophisticated mode of action
demonstrated here, when fully elucidated, may give more exploitable
leads which are needed not only in subsistence agriculture but also to
answer future world demands in agricultural production and in developing
new approaches for molecular biology in Striga. RAPD,
SSR and AFLP markers linked to genes conferring resistance to angular
leaf spot in common bean G. Mahuku, C. Montoya, Y. Mantilla, M.
Contreras, C. Jara & S. Beebe Angular leaf spot of common bean, caused by the fungus, Phaeoisaiopsis
griseola Sacc., is a major disease that can cause yield losses as
high as 80%. Host resistance is the most practical strategy to manage
the disease but resistant cultivars are short-lived because P.
griseola is highly variable. Pyramiding resistance genes is a very
effective way to develop cultivars with lasting resistance but combining
several resistance genes simultaneously in one background is impossible
without markers (either physical or molecular) for each gene. The
objectives of this study were to determine the inheritance of angular
leaf spot resistance and identify random amplified fragment length
polymrphism (RAPDs), simple sequence repeats (SSR) and amplified
fragment length polymorphism (AFLP) markers linked to the resistance
genes present in three genotypes: Mexico 54, MAR 1 and G 10474.
Greenhouse evaluations of parents, F1, F2 and backcross-derived plants
showed that ALS resistance in Mexico 54 to race 31-55 and of G 10474 to
race 63-63 was conditioned by a single dominant gene. Evaluation of
parents, F2 and recombinant inbred lines (RILs) derived from crossing
MAR1 x VAX 6 revealed that ALS resistance in MAR 1 to races 31-55, 63-23
and 31-39 was conditioned by a single dominant gene. The gene in Mexico
54 was tightly linked to the RAPD marker OPE4700 and three
potential AFLP markers E-AAG/M-CAA330,
E-ACG/M-CTC310 and E-ACT/M-CAT260. The resistance
gene in MAR 1 segregated with the SSR marker (GT)n 230,
while three AFLP fragments E-AAC/M-CAG310, E-ACA/M-CTT330 and E-AAC/M-CAT285
segregated with the resistance gene in G 10474. The utility of these markers in different common bean
backgrounds, development of sequence characterised amplified region (SCAR) markers and their usefulness in marker assisted selection in breeding to
pyramid angular leaf spot resistance genes into commercial types of
common bean is discussed. Structural
analysis of transgene rearrangements and effects on expression in
transgenic maize plants generated by particle bombardment L.
Mehlo We
co-transformed maize embryogenic callus with two plasmids, one carrying
the linked markers bar and gusA,
and the other carrying the Bacillus
thuringiensis cry1Ab gene. Molecular analysis of regenerated
transgenic plants revealed a diverse range of transgene structures,
including intact, truncated and internally-rearranged copies, and copies
interspersed with genomic DNA. We found rearrangements associated with
both functional and nonfunctional transgenes, and rearrangements
affecting the selected marker gene bar as well as the nonselected transgenes. Rearrangements often
caused loss of transgene expression, but some rearranged transgenes were
expressed normally. Conversely, while intact transgenes were often
expressed, we also detected some non-expressed copies, probably
reflecting epigenetic silencing mechanisms. All the transgenic lines we
studied contained multiple transgenes, generally comprising a mixed
array of intact and rearranged copies. Rearrangement is rarely given
serious consideration as a factor contributing to transgene silencing
and variable transgene expression, but our data show that undetected
rearrangements may have a major effect on transgene expression. This has
significant implications, especially in studies attempting to correlate
transgene copy number and expression levels and long-term gene
expression. Genetic
analysis of resistance to Striga
hermonthica introgressed from Zea
diploperennis to tropical maize A. Menkir,
A. Melake-Berhan, J.G. Kling & O. Ibikunle Striga
is the single most severe biological constraint to cereal production in
sub-Saharan Africa. Maize is highly susceptible to Striga
and can sustain yield losses of up to 100% in highly infested
fields. Host plant resistance is regarded as the most feasible and
potentially durable method to reduce losses to Striga.
In the 1980’s, IITA developed inbred lines and hybrids with partial
resistance and/or tolerance to Striga
hermonthica. However, none of
the lines and hybrids completely prevented the successful development of
Striga. Also, the level of
genetic variation resistance is too low to permit adequate progress from
selection. It was, therefore, necessary to search for novel resistance
genes that suppress the emergence of Striga
in diverse sources of maize germplsm. An accession of teosinte, Zea diploperennis, that supported little or no emergence of S.
hermonthica was identified at IITA and crossed to adapted tropical
maize. Several lines supporting few emerged Striga
plants were derived from a backcross population of Z.
diploperennis after two generations of inbreeding and repeated
selection under artificial infestation in the field and screen house.
The purpose of the study presented in this report was to use molecular
markers to identify genetic loci associated with resistance to Striga
in maize using F2:3 families derived from a cross of a Striga resistant line derived from Z. diploperennis BC4 population with a Striga-susceptible inbred line. The F2:3 families along
with the parents were evaluated in three environments under artificial
infestation with Striga and
genotyped with 47 SSR and 80 AFLP markers suing standard protocols. The
mapping population exhibited significant differences among families for Striga
damage symptom rating, numbers of emerged Striga
plants, numbers of ears at harvest and grain yield under Striga
infestation. Analyses of genotypic and phenotypic data led to the
identification of SSR and AFLP markers associated with QTLs affecting Striga
damage symptom rating, the numbers of ears at harvest and grain yield
under Striga infestation. These QTLs were consistently expressed across
test environments representing different Striga
populations and were anchored at a few common marker intervals in the
same linkage groups. Although some QTLs were significantly associated
with the numbers of emerged Striga
plants, they were not consistent across environments. The markers with
significant association to reduced Striga
damage symptom and increased number of ears at harvest and grain yield
can be used as potential candidate markers for simultaneous improvement
of these traits in maize. Future efforts should focus on search for
markers consistently linked to the number of emerged Striga
plants across environments to facilitate marker-assisted selection to
drastically deplete the reservoir of Striga
seeds from the soil. |
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