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Genetic transformation of Russian wheat varieties for the biotic and abiotic stress resistance

Dmitry Miroshnichenko, Mikhail Filippov and Sergey Dolgov.

Station ‘Biotron’, Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia

Abstract

The expression and inheritance of transgenic phenotypes were determined in T1 and T2 populations of progeny obtained after self-pollination of primary transgenic wheat (T0). A total of 16 Andros and 4 Noris T0 independent transgenic lines containing the bar/ gfp produced by microprojectile bombardment were analyzed. The inheritance of PPT-resistance and GFP expression generally followed a Mendelian inheritance pattern for a single, dominant locus. Most transgenic progeny showed resistance to high concentrations of herbicide (up to 2.5% Basta). However transgene silencing, distorted transgene segregation, low herbicide resistance and the absence of the GFP fluorescence were observed in the progeny of two transgenic lines. In general, no clear difference in the herbicide resistance level was found between homozygous and heterozygous transgenic T1 plants. To enhance fungal resistance a number of transgenic wheat lines with pathogen-related genes like rice thaumatin-like protein TLP and oat permatinI were generated.

Media summary

The inheritance and expression of herbicide resistance gene bar was assessed in T1 and T2 populations of 22 transgenic lines of two Russian wheat varieties.

Key Words

Wheat, herbicide resistance, genetic transformation, transgene inheritance

Introduction

Wheat (Triticum aestivum L.) ranks first among cereal crops cultivated in Russia, 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, transferring genes conferring these traits is limited as they are mostly quantitative traits. Nonetheless, the direct introduction of a small number of genes by genetic engineering offers a convenient and rapid approach for the improvement of stress tolerance. Weeds compete with crop plants for available nutrients and light energy, and thus reduce crop yields by an average of 10 to 15%. Through biotechnology, it has been possible to provide crop plants with herbicide resistance, which allows selective elimination of weeds. The biolistic transformation approach currently is the best choice for generating useful transgenic wheat germplasm in a genotype-independent manner. Efficient regeneration protocols have been developed at the ‘Biotron’ for a number of Russian spring and winter cultivars. Using explants at an optimal embryogenesis stage we have improved the efficiency of biolistic transformation. Moreover, the combination of gfp as a vital reporter gene and bar genes for transgene selection allowed the development of efficient transformation protocols for elite Russian wheat cultivars (Miroshnichenko et al., 2003). In this report the stability and heritability of herbicide resistance gene (bar) was investigated in heterozygous- and homozygous populations of a number of independent transgenic wheat lines.

Methods

The monocot transformation vector psGFP-BAR (Richards et al. 2001) was used to generate herbicide-resistant wheat plants. It 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). Two plasmids were used to investigate expression of PR-related genes: psGFP-BAR-D34 and psUbiOP. Both plasmids are derivative from psGFP-BAR. Plasmids psGFP-BAR-D34 contains a 3.0 kb HindIII fragment from pCamBar-UbiTLP (Velazhahan et al. 1998) including the coding sequence of rice TLP-D34 gene driven by the maize ubiquitin Ubi1 promoter. The second plasmid psUbiOP contains 846 bp EcoRI fragment of Oatperm1 gene coding sequence (kindly provided by Dr. R.Skadsen; Skadsen et al. 2000) driven by the Ubi1 promoter. A particle inflow gun was used to deliver DNA-coated tungsten particles into the immature embryos. Expression of gfp gene in wheat tissues was visualized using a fluorescence stereomicroscope Opton ICM 405. All plants were screened for the presence of transgene sequences by PCR. Approximately 18-20 days after anthesis, the developing T1 progeny seeds of greenhouse grown T0 primary transgenic lines were removed, surface-sterilized and germinated on MS basal medium to observe GFP expression (Fig. 1.A.). T1 embryos were geminated without PPT selection to quickly obtain T1 plants. The number of examined T1 embryos varied from 15 to 95 for each independent T0 transgenic line. To study the herbicide resistance of transgenic T1 heterozygous- and homozygous populations, herbicide (Basta 2.5 %) was applied by ‘painting’ the distal, upper halves of leaf surfaces with cotton buds. Two leaves from the one T1 plants were treated. Resistance was measured 7 days post-application. Plants were distributed into 6 groups according leaf damage (Figure 1.C.). All T1 plants that were expressing herbicide resistance were further assessed for homozygous or heterozygous inheritance of gfp/bar genes. The 18-20 day-old immature T2 embryos were cultured on MS medium supplemented with 10 mg/l PPT and 0.1 mg/l GA3 to quickly obtain T2 plants and to rapidly identify homozygous transgenic plants among them. The embryos from nontransformed Andros or Noris plants did not germinate on this selection medium. After 5 days in culture, most of the transgenic seedlings were longer than 5 cm. Homozygous T2 wheat seedlings were transferred to the greenhouse and were assessed for Basta resistance according the methods applied for T1 transgenic plants.

Results

Using immature zygotic embryos as the target tissue for bombardment we have generated 53 independent transgenic lines of two Russian spring wheat varieties Andros and Noris. T1 progeny from 16 randomly chosen independent transgenic T0 lines of Andros and from 4 independent T0 lines of Noris were studied for stability of transgene inheritance and gfp/bar expression. Both Mendelian and non-Mendelian inheritance of GFP expression was found. The majority of lines 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 (Table 1). After germination most of GFP positive transgenic T1 seedlings were transferred to the greenhouse and grown to maturity to perform herbicide resistance tests. Prior to analysis all T1 plants were assayed for bar gene inheritance by PCR analysis. All selected GFP positive T1 seedlings showed the presence of bar gene sequence in the genomic DNA. None of the GPP negative segregants had the bar gene sequence in their genome.

In an initial sensitivity test to determine a suitable Basta concentration for the assay, leaf segments of T1 GFP negative plants and non-transgenic plants of Andros and Noris showed no significant tolerance to 1% Basta (Fig. 1.B.), while PCR positive plants were capable of tolerating up to 3% concentration. Application of 2.5% Basta to sections of nontransgenic leaves resulted in necrosis of the treated leaf sections within 3 to 5 days. Early symptoms of damage were chlorosis at the end of the second day after treatment, followed by a loss of turgor, complete chlorosis, and total desiccation of at least part of the treated tissue within a week of application. Transgenic plants expressing herbicide resistance, when treated with 2.5% Basta, were distributed into 6 groups according leaf damage, ranging from no detectable symptoms to a total leaf chlorosis and desiccation (Fig. 1.C). Four lines showed a high resistance to 2.5 % Basta, as the average resistance of A 27, A 42, A 48 and N 6 was more that 4 (Table 1). In most lines resistance to herbicide fluctuated greatly from plant to plant. Only the progeny of one Noris transgenic line (N 4) had very low resistance to herbicide, suggesting bar was not expressed. Such loss of herbicide resistance was reported earlier in many crops generated by particle bombardment.

All T1 wheat plants showing Basta resitance were assessed for homozygous or heterozygous inheritance of gfp/bar genes. The data from the germination tests indicated that part of T2 plants did not segregate for PPT sensitivity and were homozygous for the bar gene. Most of PPT resistant seedlings showed GFP fluorescence (Fig. 1.A). The only exception was found between T2 embryos of the N6 line: only the half of the cultured embryos showed gfp expression. However PCR analysis confirmed the presence of gfp gene in all T2 PPT+GFP- plants from the N4 line. It should be noted that overall level of GFP expression in the embryos of this line was lower than in the T2 transgenic embryos of other lines.

The identification of transgene inheritance allowed us to compare the herbicide resistance level between hetero-or homozygous T1 plants. To date there have been only limited studies in cereals to examine the difference in transgene expression between homozygous and heterozygous transgenic plants. In some reports the homozygous state of plants positively influence the expression of transgene. Baruah-Wolff et al. (1999) observed a subset of transgenic T1 rice plants that expressed the firefly luciferase gene at high levels were scored as homozygous. High levels of transgenic protein have also been associated with homozygoity of the transgene in rice (Duan et al. 1996). On the other hand no difference in transgene expression was detected between homozygous and heterozygous rice plants carrying the gusA gene (Peng et al. 1995) or maize plants expressing the cryIA(b) gene (Fearing et al. 1997). James et al. (2002) found that the level of gusA expression between homozygous and heterozygous T2 rice plants was influenced by the studied transgenic line. They found both negative and positive influence of the homozygous state of plants on the average GUS activity. 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 (A17 and N6) had the higher herbicide resistance associated with homozygity of the transgene However, further studies on a wider range of homologous and heterologous transgenes are needed to understand the stability of herbicide resistance in wheat.

More than 100 homozygous T2 progeny with gfp and bar genes expression from transgenic T1 plants were grown in the greenhouse. To date, the herbicide-resistance test was carried out with the T2 population of 12 T1 homozygous plants belonging to 5 independent wheat lines. This test revealed the high degree of variation between the T2 homozygous populations belonging to one line. However, there was much less variation between T2 progeny and their T1 parent. Although the results of this study do not address the underlying molecular phenomena, the observed inheritance and expression patterns provide important empirical information that will be useful in designing breeding approaches.

Table 1. Analysis of Basta ( 2.5 %) resistance in transgenic population of T1 plants produced by self-pollination of independent transgenic wheat lines transformed with psGFP-BAR.

Transgeenic .line

Segregation

Number of T1

Average resistance#

   

plants tested

Heterozygous T1 plant

Homozygous T1 plant

Mean for the line

A3

15 : 1

10

2.60

3.20

2.90

A6

3 : 1

10

2.90

3.40

3.00

A15

3 : 1

17

2.95

3.18

3.05

A16

3 : 1

11

4.13

3.54

3.82

A17

3 : 1

19

2.47

4.25

2.84

A18

3 : 1

20

3.30

3.38

3.15

A20

3 : 1

35

2.65

3.17

2.83

A27

3 : 1

7

4.25

3.67

4.00

A34

3 : 1

10

3.00

3.67

3.20

A37

3 : 1

15

3.60

3.20

3.47

A39

3 : 1

10

2.50

n.f.*

2.50

A40

15 : 1

15

2.92

3.00

2.93

A42

3 : 1

18

3.91

4.57

4.17

A47

3 : 1

20

3.26

4.00

3.30

A48

1 : 1

9

4.25

4.80

4.33

A53

3 : 1

10

3.22

3.00

3.20

N2

3 : 1

7

3.71

n.f.*

3.71

N4

3 : 1

15

0.54

2.00

0.73

N6

3 : 1

11

4.60

4.00

4.55

N9

3 : 1

12

3.33

n.f.*

3.33

* homozygous T1 plants were not found in the T0 lines.
# On an arbitrary scale 0-5; see Fig 1 for details.

Fungal wheat diseases are 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 increase resistance to Botrytis cynerea. This suggests that the approach can contribute to quantitative resistance. In this study we report the introduction of several antifungal genes in wheat plants. 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 5 independent T0 transgenic lines of Andros have been generated with psGFP-BAR-D34 and psUbiOP, respectively. The presence of TLP and oatpermI sequences were found in the genome of all T0 transgenic plants grown in the greenhouse. More than 160 T1 plants obtained after self-pollination of 8 independent TLP-expressing T0 lines. T1 plants were selected upon expression of gfp gene in the zygotic embryos of T0 lines. Currently over 30 lines homozygous for the TLP gene T1 plants were identified. The populations of homozygous plants are under investigation for resistance to a range of important wheat pathogens.

Figure 1. Expression of gfp and bar genes in the progeny of transgenic wheat transformed with psGFP-BAR construct. gfp gene expression in immature zygotic embryos of T1 progeny (on the top – embryo of non-transgenic segregant without expression, at the bottom – embryo with gfp expression). B. Basta resistance assay (1.0% Basta) on T1 progeny of A42 transgenic line of Andros, right – non-transgenic plant, left – transgenic plant. C - Herbicide damage ranging upon treatment with 2.5% Basta (0 – full death of leaf segment, 1 – more than 75% of leaf are damaged, 2 - 75-50% of leaf are damaged, 3 – 25-50% of leaf are damaged, 4 – less than 25% of leaf are damaged, 5 - no visible damage).

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