Download genetic transformation of russian wheat cultivars

GENETIC TRANSFORMATION OF RUSSIAN WHEAT CULTIVARS.
Dmitry Miroshnichenko*, Mikhail Filippov and Sergey Dolgov
‘Biotron’, Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian
Academy of Sciences, Pushchino, Moscow Region, 142290 Russia
Wheat (Triticum aestivum L.) ranks first among cereal crops cultivated in Russia and Russia is
one of the major wheat producers in the world. Though there is a great demand for Russian wheat
varieties with resistance to diseases and other environmental stress, there is a limitation at
transferring resistance to crop plants by the complexity of stress tolerance traits, as most of these
are quantitatively linked traits. Nonetheless, the direct introduction of a small number of genes by
genetic engineering offers convenient alternative and a rapid approach for the improvement of
stress tolerance.
The biolistic transformation approach currently is the best choice for generating useful transgenic
wheat germplasm in a genotype-independent manner. During last years efficient regeneration
protocols were developed at the ‘Biotron’ (Branch of Shemyakin and Ovchinnikov Institute of
Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Russia), for number of Russian
spring and winter cultivars. Using wheat explants at an optimal embryogenesis stage we have
improved the reproducibility of biloistic transformation efficiency.
Fig 1. Vectors used in experiments for wheat transformation
The monocot transformation vectors psGFP-BAR (Richards et al. 2001) and pAct1-F (McElroy
et al., 1990) were used for generation of wheat transgenic plants. First plasmid contains the
synthetic codon optimized red-shifted gfp gene (sgfpS65T) driven by the rice Act1 promoter
(McElroy et al., 1990) and bar gene, which confers resistance to the herbicide Basta (active
ingredient L-phosphinotricin [PPT]), driven by the maize ubiquitin Ubi1 promoter (Christensen
et al. 1992). Second vector contains the gus reporter gene, encoding β-glucuronidase, driven by
the rice Act1 promoter. For co-transformation experiments, a molar ratio of 1:2 GFP/bar
(selectable) to gus (nonselected) cassettes was used.
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Fig 2. Selection of transgenic plants after bombardment. A. Transient gfp expression in wheat
immature embryo B. Direct regeneration of transgenic wheat somatic embryo. C. Formation of
transgenic wheat embryogenic callus expressing gfp. D. Formation of transgenic embryos from
embryogenic callus.
The combination of gfp as vital reporter gene and bar genes for transgene selection allowed
establishing no-escape efficient protocols for elite Russian wheat cultivars. Using own optimized
transformation protocol we have generated 53 independent transgenic lines of two Russian spring
wheat varieties Andros and Noris.
Fig 3. Transgenes expression in wheat tissues. A. GUS expression in young stem/spike of
tansgenic wheat. B. gfp expression in young spike. C. gfp expression in anthers (left and center anthers of transgenic wheat, right non-transgenic).
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Inheritance and stable expression of transgenes is an important concern in crop improvement
through gene manipulations. Progeny from 22 independent transgenic lines of Andros and from 5
independent lines of Noris were studied. Isolated immature embryos were germinated in vitro,
transferred to the greenhouse and T1 seedlings grown to maturity. Segregation studies at the
expression level, using GFP fluorescence (Fig 4) followed by histochemical GUS staining (for
lines generated after co-bombardment), showed both Mendelian and non-Mendelian inheritance
of GFP and GUS expression. The majority of lines (19 out of 27) exhibited 3:1 segregation in T1
progeny indicating that active copies of bar/gfp genes had integrated at a single locus and were
inherited as a simple Mendelian trait. Eight lines deviated from this segregation and showed
unusual segregation patterns varying from 5:1 to 20:1. Such distorted ratios have been reported
previously in wheat and other plants. The causes for the unpredicted inheritance of transgenes are
unclear but gene silencing or gene instability may play a significant role. After germination most
of GFP positive transgenic T1 seedlings were transferred to the greenhouse and grown to
maturity to perform herbicide resistance tests.
Fig 4. Analysis of transgenes expression and inheritance in T1 progeny after upon one-day (A, B)
and five-days (B, D) culture on germination medium. A, C - embryos of null-segregant. B, D transgenic embryos.
The identification of transgene inheritance allowed us to compare the herbicide resistance level
between hetero-or homozygouse T1 plants. To date there are only limited studies in cereals were
carried out the difference in transgene expression between homozygous and heterozygous
transgenic plants. In some reports the homozygous state of plants positively influence on the
expression of transgene in rice (Baruah-Wolff et al, 1999; Duan et al. 1996). In the other hand no
difference in transgene expression was detected between homozygous and heterozygous rice
plants carrying the gusA gene (Peng et al. 1995; James et al. 2002) or maize plants expressing the
cryIA(b) gene (Fearing et al. 1997). In our study the comparision was performed for 17 out of 20
wheat lines. In general we observed the same level of herbicide resistance between homozygous
and heterozygous T1 wheat plants. Only two transgenic lines found to have the higher herbicide
resistance associated with the homozygous state of plants. However, further studies on a wider
range of homologous and heterologous transgenes are needed to be carrying out for an
understanding of herbicide resistance stability in wheat.
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Fig 5. Field design for testing herbicide
resistance of transgenic wheat lines. A, B, C,
D – experimental plots contained 18 subplots
with randomized distributed subplots of
transgenic (NN 1-16) and non-transgenic
control (A, H). Subplots B and C were
treated with 1% Basta.
Fig 6. Fields plots before treatment with
herbicide (A) and before the harvest (B)
Evaluation of transgenic plants under field conditions is necessary to determine the effect that
genetic transformation could have on the agronomic traits of crops. However, the nature of some
traits, like yield and the content of biomass, was very influenced by the environment, and the fact
that the transgenic plants are obtained by an in vitro culture process make these traits difficult for
evaluation. In 2004 we have performed field evaluation of the efficiency of T3 homozygous
plants from seven transgenic lines of Andros and Noris for herbicide resistance. Transgenic and
non-transgenic seeds were sown in four plots divided onto 18 subplots (Fig 5, 6). Subplots were
representing one of 16 transgenic T3 families and two controls. Two plots were testes for
herbicide resistance; other two plots were served as control and were not treated with herbicide.
Young developing plants were treated with 1% Basta. One week latter non-transgenic wheat
plants showed no sings of resistance, while most transgenic plants exposed high level of
herbicide resistance (Fig 7).
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Fig 7. Fields trials for herbicide resistance of transgenic wheat lines (T3 progeny) upon treatment
with 1% Basta. A. before treatment. B. one week after treatment.
To determine the effect of herbicide treatment, several traits such as plant height, the weight of
seeds in one spike, the number of spikes per m2 and overall yield were evaluated at the end of
wheat cultivation. The result of this experiment showed that yield-data of transgenic and control
plants were comparable. Althought two of 16 transgenic T3 families had a clear difference with
non-trangenic control. It should be noted that all variant (treated and non treated) of those two
families resulted in similar loss of yield and decreasing of plants height (Fig 8), thus indicating
the somaclonal nature of such deviations. The difference between non-treated transgenic/nontransgenic plants and treated transgenic plants was not statistically proved, though certain loss
and gain in yield was observed between transgenic lines due to environmental influence.
Although the results of this study do not address the underlying molecular phenomena, the
observed expression patterns provide important empirical information that will be useful in
designing breeding approaches for stress resistance. Additionally it was the first attempt of filed
evaluation of transgenic wheat in Russia.
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Fig 8. The difference between two transgenic lines (a, b) in plant height, due to somaclonal
variations.
Fungal wheat diseases is the major biotic factor that limits wheat productivity in Russia, thereby
causing enormous loss. Control of fungal wheat diseases by fungicide application and other
practices is neither practical nor sustainable. Previously, we have shown that constitutive
expression of thaumatin II (belong to PR-5 group) in strawberry can indeed plant increase
resistance to Botrytis cynerea. This suggests that the approach can contribute at least to
quantitative resistance. To enhance fungal resistance of Russian wheat cultivars we are working
on the over expression of several pathogen-related genes like thaumatin-like protein TLP (from
rice) and oatpermI (from oat). Currently 12 and 9 independent T0 transgenic Andros lines have
been generated with genes of those PR proteins. The presence of TLP and outperm sequences
was found in the genome of all T0 transgenic plants grown in the greenhouse. More than 160 T1
plants obtained after self-pollination of independent TLP-expressing T0 lines. Currently over 30
homozygous for TLP gene T1 plants were identified. The populations of homozygous plants
currently are under investigation for the resistance to main injurious wheat phatogenes.
Fig 9. Authors (Miroshnichenko and Fillipov) on the experimental field plot in august 2004
before the harvest and drying of wheat spikes.
Another problem for productivity of cereal crops including wheat is soil salinity. This problem is
existed long before humans and agriculture, but the problem has been aggravated by agricultural
practices such as irrigation. Today, ~20% of the world’s cultivated land and nearly half of all
irrigated lands are affected by salinity. Thus, if salt tolerance can be conferred upon wheat plants,
the production of wheat could be increased. Recently, genes encoding vacuole-type Na+ /H+
antiporters have been shown to increase the salinity resistance of several transgenic species
including arabidopsis, brassica, tomato and rice. We are currently working on the production of
salt- resistant wheat plants using Na+ /H+ antiporter gene HNHX isolated from barley genome by
the research group of prof. Babakov A.V. (All Russian Institute of Agricultural Biotechnology,
RAAS). To date a number of morphologically normal and fertile transgenic T0 plants have been
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generated. Resistance/susceptibility to the high soil concentrations is planed to be investigated in
the end of 2005 after generation of homozygous T3 and T4 transgenic progeny.
Source:
Black Sea Biotechnology Association
www.bsbanet.org
March 2005
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