55. Pyramiding agronomic traits in transgenic plants
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P.
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TINJUANOJUN’, S. B. MAQBOOL’, D. GAHAKAWA’, L. MEHLO’, D.
Sudhakar’, T. N. Loc2,
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A. Kouu’, A. M. R. GATEHOUSE2, J. A. GATEHOUSE~ and P. CHRISTOU’
1) Molecular Biotechnology Unit, John Innes Center, Colney
Lane, Norwich NR4 7UH, UK
2) Department of Biological Sciences, University of Durham,
Durham DH1 3LE, UK
Pests and diseases cause the loss of up to 30% of the world’s crop harvests, despite breeding programs that enhance resistance by exploiting endogenous resistance genes, and the extensive use of chemical pesticides. Genetic engineering can complement and accelerate breeding programs by introducing resistance genes from diverse sources, such as other plants, bacteria and even animals. There are, however, a number of problems associated with such genetic engineering approaches to crop improvement. Firstly, it is necessary to develop efficient transformation and regeneration protocols. Unfortunately, transformation and regeneration efficiencies are strongly genotype dependent in most crop plants, so that it may be relatively easy to obtain transgenic plants of certain model varieties but not those of commercial importance. Secondly, once transgenic plants have been produced, there is the possibility that pests may evolve resistance to the transgenes used against them, rendering the transgenic plants no less susceptible to pest attack than the wild type.
In this communication, we first
present data showing how we have optimized particle bombardment for the
transformation of many traditionally recalcitrant rice genotypes, and report
the first successful transformation of some notable commercial varieties
such as KDML1O5 and SP6O. We then report success with two strategies aiming
to prevent or delay the evolution of resistance in insect populations targeted
by insecticidal transgenic plants: transgene pyramiding and toxin augmentation.
Kaho Dawk Mali 105 (KDML 105) and
Supanburi 60 (SP6O) are two commercially important Thai rice varieties
representing a major sector of the Thai rice export market. Both varieties
are susceptible to insect pests, including the brown planthopper (BPH)
which causes direct damage to crops through its feeding mechanism (phloem
abstraction) and also acts as the vector for several rice viral diseases.
Homopteran insects like BPH are difficult to control since they are not
targeted by any known Bt cry gene, and because their feeding method causes
them to avoid the ingestion of topical insecticides. However, a lectin
isolated from snowdrop (GNA; Galanthus nivalis agglutinin) has been shown
to cause extensive insect mortality, reduced fecundity and developmental
arrest as well as displaying potent antifeedant activity. The gna gene
has been introduced into a number of model rice genotypes and its efficiency
in the control of BPH and other sap-sucking insects has been demonstrated
(Rao et al. 1998; Sudhakar el a!. 1998). Recently, we have produced the
first ever transgenic KDML1O5 and SP6O rice plants containing this gene,
and insect bioassays have confirmed both antifeedant and insecticidal properties
in the transgenic plants. We used three transformation plasmids containing
the gna gene: one in which the gene was driven by the maize ubiquitin-
1 promoter, one in which it was driven by the phloem-specific rice sucrose
synthase-l promoter, and a third cointegrate vector containing gna driven
by the ubiquitin-l promoter linked to the selectable marker hpt. We found
that immature rice embryos were the most suitable explants for transformation.
The successful production of transgenic plants of two cultivars traditionally
regarded as recalcitrant to both transformation and regeneration opens
the way for introducing agronomically useful traits into any commercially
grown variety (Fig. 1). Ultimately, this will provide the best opportunity
for maximizing yields throughout the world, since it should be possible
to engineer transgenic plants of any variety, suitable for farming in any
region of the world.
Once transgenic plants have been
generated, it is important to avoid the evolution of resistance in target
pest populations. Resistance has evolved to single insecticidal transgenes
both in laboratory tests and in field populations, because a single corresponding
mutation is all that is required in the targeted insects. Strategies developed
to avoid this outcome in large, commercially-grown transgenic crops include
Refugia (the provision of spatial or temporal refuges containing nontransgenic
plants) and the use of regulated promoters (to restrict insecticidal transgene
expression to certain plant regions or critical developmental stages).
Both strategies reduce selection pressure to encourage the survival and
inter-breeding of nonresistant insects. Alternative strategies seek to
avoid the evolution of ‘tit-for-tat’ resistance by increasing the range
of molecular targets for the toxin in the susceptible insect pest. Transgene
pyramiding involves the simultaneous expression of multiple transgenes
active against different molecular targets in the same pest, so that single
mutations in the susceptible insects would no longer be sufficient to establish
resistance, We have generated transgenic plants simultaneously expressing
the Bt genes cry1Ac and cry2A, which both target lepidopteran pests such
as the rice leaf folder. The same plants also contained the gna transgene,
and thus showed broad spectrum resistance to multiple insect pests with
pyramiding of Bt genes to avoid or delay the evolution of resistance against
single Bt toxins (Fig. 2). Western blots showed the combined expression
of all three insecticidal transgenes and bioassays confirmed the effectiveness
of the transgenic plants against several pests: the rice leaf folder, yellow
stemborer and brown planthopper (Maqbool and Christou 1999)
Furthermore, we have conducted studies
to show that the behavior of such multitransgene loci is similar in a diverse
range of rice genotypes. We selected germplasm representing 11 diverse,
commercially-grown rice cultivars and simultaneously transformed rice tissue
with two insecticidal transgenes and three markers. We investigated transgene
expression levels, locus structure, and the stability of transgene expression
over four generations. We found that different genotypes behaved in a similar
fashion, and that stable long term expression of multiple transgenes was
a reality (Gahakwa et a!. in press). Hence, it should be possible to devise
and optimize a suitable multi-trait and or pyramiding strategy in any rice
genotype and transfer the same technology to any other genetic background,
providing maximum protection for farmers throughout the world.
Finally, we report an entirely novel
strategy to prevent or delay the evolution of resistance. In this case,
we have used recombinant DNA techniques to add a novel binding domain to
the Bt toxins CrylAb and CrylAc. We chose the galactose-binding domain
of the ricin toxin B chain, which can bind to any galactose-containing
oligosaccharide. We have produced recombinant toxins in cultured insect
cells using the baculovirus expression system. The fusion proteins showed
strongly enhanced toxicity towards insect cells compared to the unmodified
Bt toxins alone. Furthermore, we have generated transgenic plants containing
the same fusion constructs and shown elevated toxicity against BPH larvae
in bioassays. This recombinant DNA approach provides yet another strategy
to make a pre-emptive strike in the arms race between insects and plants,
and tip the balance firmly in the favor of rice growers.
References
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and A. Kohli, 1999. Transgenic rice as a system to study the stability
of transgene expression: multiple heterologous transgenes show similar
behaviour in diverse genetic backgrounds. Theor. AppI. Genet. (in press).
Maqbool, S.B. and P. Christou, 1999. Multiple traits of agronomic
importance in transgenic indica rice plants:
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