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. 2016 Oct 20;7(3-4):159–174. doi: 10.1080/21645698.2016.1240148

Molecular and functional characterization of cry1Ac transgenic pea lines

Alemayehu Teressa Negawo a,b, Linda Baranek a, Hans-Jörg Jacobsen a, Fathi Hassan a,c,✉
PMCID: PMC5161004  PMID: 27764552

ABSTRACT

Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

KEYWORDS: cry1Ac protein, feeding damage, insect pests, larval mortality, Pea, transgenic resistance

Introduction

Insect pests are biotic factors challenging production of food and thus threaten the global food security. Estimated yield losses due to insect pests vary from crop to crop and region to region. Sharma et al., 2010 In pea, 10 to 70 % yield losses can be incurred depending on the insect pests. Schroeder et al., 1995; Clement, Hardie, Elberson 2002; Legowski, Gould 1960; Williams, Schotzko, Okeeffe 1995; Biddle, Cattlin 2001; Korth 2008

When a desired trait is not available in a given gene pool, one way to complement conventional breeding is to apply biotechnological techniques to access and transfer genes for novel traits from other sources. This approach has been used to develop not only varieties resistant to production constraints (such as insect and diseases) but also to improve the nutritional value of different crops.Korth, Stewart 2008 Pea is one of the economically important legume crops that have been extensively studied under in vitro conditions during the last few decades. Today, against the odds of many grain legumes which are recalcitrant to in vitro conditions, there is a well established and routinely used regeneration and transformation protocol Schroeder et al., 1993 for pea. Transgenic pea lines against different production constraints such as insect pests and diseases have been developed.Schroeder et al., 1995; Shade et al., 1994; Hassan, Meens, Jacobsen, Kiesecker et al., 2009; Richter 2006

Despite the successful application of transgenic approaches in pea, little attention has been given to insect resistance development using cry genes from Bacillus thuringiensis (Bt). The Cry proteins are targeted to the larval stage of the insect, they are activated in the midgut of larvae and bind to the receptors on the epithelial cell membrane leading to the formation of membrane pores. The formation of lytic pores causes the uncontrolled release of ions, the collapse of the epithelia that stops the larvae from feeding and finally leads to the death of the larvae. Bravo et al., 2011 In line with this gap, we have reported the successful development of transgenic pea lines expressing cry1Ac gene from Bacillus thuringiensis. Negawo et al., 2013 The cry1Ac gene Cheng, Sardana, Kaplan, Altosaar 1998; Sardana 1996 is one of the commonly used Bt cry genes to develop transgenic lines in many plant species such as tobacco, cotton, maize, soybean, tomato and rice, Cheng, Sardana, Kaplan, Altosaar 1998; Perlak 1990; Bohorova 1999; Stewart Jr CN 1996; Mandaokar et al., 2000 to become resistant to Lepidopteran pests. Hofte, Whiteley 1998; Crickmore 1998

Lepidopteran insect pests belong to the major pests of pea and many other legume species as they cause significant yield losses under field conditions. Sharma et al., 2010 Their control by conventional measures is inefficient due to the absence of resistant varieties. Even though chemical pesticides are used at high costs, insect pests cause an average yield loss of 30 % in legumes. Sharma et al., 2010 Sometimes, it is difficult to target the insect pests (e.g. larval stage) via pesticide application since they are located inside the crop tissue or the seeds. Christou, Twyman 2004 There is also a clear socio-environmental concern related to the heavy use of chemical pesticides in agriculture production system, Sharma et al., 2000 which needs to be changed. In addition, not many insecticides are approved for pea. Besides an enormous number of chronic illnesses related to pesticide poisoning, about 200,000 people are estimated to die yearly worldwide due to improper application of pesticides. Odukkathil, Vasudevan 2013 Hence, the application of transgenic approaches is a viable alternative strategy to control the insect damage and reduce the load of chemical pesticide in the agricultural system Christou, Twyman 2004 and finally increase the crop yield available for consumers.

Thus, the current study describes the development of insect resistant pea plants by analyzing cry1Ac transgenic pea lines at molecular and functional levels.

Results

PCR Detection of Cry1Ac Gene in the Genome of Transgenic Pea Lines

The PCR analysis confirmed the genomic integration of the cry1Ac transgene in the analyzed transgenic lines (Fig. 1). Up to T2-T7 advanced generations were obtained for some of the transgenic lines indicating the inheritance of the introduced transgene to the next generation.

Figure 1.

Figure 1.

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PCR detection of transgene genomic integration and inheritance to the next generation in some of the transgenic lines. Transgenic pea lines and their subsequent generations were analyzed using primers for cry1Ac (750 bp) and HMG-I/Y (570 bp) genes indicating the stable genomic integration and inheritance of the cry1Ac transgene. L: GeneRuler™ 100 bp plus DNA ladder, +C: plasmid (pGII35S-cry1Ac) DNA as a positive control, -C: genomic DNA from non-transgenic pea plant as a negative control, W: water control; and DA, B3, D2R and C1 are different transgenic pea lines.

RT-PCR Detection of Transgene Expression in the Transgenic Pea Plants

To analyze the expression of the cry1Ac gene at the transcriptional level, the synthesis of cDNA from isolated RNA was done using the Revert Aid First Strand cDNA synthesis kit (Thermo Scientific). PCR amplification using only RNA samples showed the absence of genomic DNA contamination in the isolated total RNA. The prepared cDNA was used in the PCR detection of the cry1Ac gene specific sequence. The expected PCR products for both cry1Ac and HMG-I/Y housekeeping genes were amplified in most of the analyzed plants (Fig. 2, Table 1). Out of 58 plants (T2-T7 generations) analyzed from 15 transgenic lines, 44 plants from 13 transgenic lines showed the expression of cry1Ac at the transcriptional level.

Figure 2.

Figure 2.

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RT-PCR expression analysis of cry1Ac transgene (750 bp) and pea HMG-I/Y housekeeping gene (570 bp from genomic DNA and 350 bp from cDNA) in the advanced progenies of different transgenic pea lines. L: GeneRuler™ 100 bp plus DNA ladder, +C: plasmid (pGII35S-cry1Ac) DNA as a positive control, -C: genomic DNA of non-transgenic pea plant as a negative control; W: water control, lane 1-12 and 14-20: cDNA from different transgenic lines and lane 13: cDNA from non-transgenic control plant [1: D2R-1(T1), 2: E15(a)-2-1(T2), 3: G/A-1-1(T2), 4: BR-5-1-4-1(T4), 5: DA-2-3-6-1(T4), 6: DqR-8-7-2-1(T4), 7: C1-2-6-15-1(T4), 8: G51-2-2-3-1-1(T5), 9: C1-2-3-3-3-2-1(T6), 10: C1-2-6-13-1-3-6-1(T7),11: B1-2-1(T2), 12: G51-1-5-1(T3), 14: B3-1-11-4-1(T4), 15: G51-1-11-1(T3), 16: A2/D12-1-1-1-1(T4), 17: A2R-2-1(T2), 18: G51-1-5-1 (T3), 19: D2R-2-12-1(T3) and 20: B1-2-1(T2)].

Table 1.

Summary of RT-PCR expression analysis of cry1Ac gene in the transgenic pea lines.

Plant code Progeny level Cry1AcPCR RT-PCR Plant code Progeny level Cry1AcPCR RT-PCR
A2/D12 T0 + + C5-3-1 T2 + −
A2/D12-1-3 T2 + + C7 T0 + +
A2/D12-1-4 T2 + − C7-1-3-1 T3 + −
A2/D12-1-1-1-1 T4 + − C7-1-4-1 T3 + −
A2R-2-1 T2 + − C7-1-4-3 T3 + −
B1 T0 + + D T0 + +
B1-1-1 T2 + + D20 T0 + +
B1-1-2 T2 + − D2R T0 + +
B1-1-3 T2 + − D2R-1 T1 + +
B1-2-1 T2 + + D2R-2-9-5 T3 + +
B2 T0 + + D2R-2-12-1 T3 + +
B2R T0 + + DA T0 + +
B3 T0 + + DA-2-1 T2 + +
B3-1-4-2 T3 + + DA-2-2 T2 + −
B3-1-11-4-1 T4 + − DA-2-3 T2 + +
B3-3-1 T2 + + DA-2-3-6-2 T4 + +
B3-3-1-1 T3 − − DA-2-4 T2 + +
B3-3-1-2 T3 + + DqR T0 + +
B3-3-1-4 T3 + + DqR-8-1 T2 + +
B3-3-1-5 T3 + + DqR-8-2 T2 + +
B3-3-2-1 T3 + + DqR-8-4 T2 + +
BR T0 + + DqR-8-7-2-1 T4 + +
BR-3-1 T2 + + DqR-8-8 T2 + +
BR-5-1 T2 + + DR T0 + +
BR-5-1-4-1 T4 + + DT T0 + +
BR-5-2 T2 + + E T0 + +
C1-1-2 T2 + + E1 5(a)-2-1 T2 + +
C1-2-1-6-13-1 T5 + + ER T0 + +
C1-2-1-6-13-2 T5 + + G T0 + +
C1-2-1-6-13-3 T5 + + G/A-1-1 T2 + +
C1-2-1-6-13-5 T5 + + G3 T0 + +
C1-2-3-3-3 T4 + + G4 T0 + +
C1-2-3-3-3-2-1 T6 + + G51-1-5-1 T3 + +
C1-2-3-6-2 T4 + + G51-1-11-1 T3 + +
C1-2-6-13-1-3-6-1 T7 + + G51-2-2-3 T3 + +
C1-2-6-15-1 T4 + + G51-2-2-3-1-1 T5 + +
C5-1 T1 + + GB T0 + +
C5-1-1 T2 + − GBR T0 + +
C5-2-1 T2 + − Positive Control NA + NA
C5-2-2-1 T3 + − Negative Control NA − −
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Expression Level of Cry1Ac Transgene in the Transgenic Pea Lines

The expression levels of the cry1Ac gene were determined using qRT-PCR for some selected lines. The melting curve analysis of the amplification products showed a single peak on each line of the curve indicating the specificity of the product in each reaction (Fig. 3). The normalized (using reference gene, HMG) expression folds vary from line to line ranging from very low (< 0.1) to 4.72 folds (Fig. 4). The highest expression level (4.72 folds) was observed in A2/D12-1-1-1-1 plant followed by BR-5-1-4-1 (2.85 folds) and C1-2-6-13-1 (2.62 folds) plants. Eleven of the analyzed plants showed expression folds of more than one. The rest of the analyzed plants showed expression folds of less than one with 8 plants showing very low expression level (< 0.1 folds).

Figure 3.

Figure 3.

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The melting curve analysis of the amplification products produced during qRT-PCR analysis. The graphs show the negative derivative of fluorescence accumulation plotted against the temperature (°C) for each reaction. The presence of a single peak on each line of the curve shows the absence of unspecific PCR product for each reaction.

Figure 4.

Figure 4.

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Expression levels of cry1Ac transgene in different transgenic pea lines. Normalized fold expression data represent the mean of 3 replicates (Mean ± SE).

Detection of Cry1Ac Protein in the Transgenic Pea Lines

The accumulation of the Cry1Ac protein in the leaves of different lines was detected using a commercial detection strip specific for Cry1Ab/Cry1Ac proteins. The expected test signal for the Cry1Ac protein was observed in the protein extract of most of the transgenic plants. On the other hand, the test signal was not developed on the immunostrip placed in the protein extract of control and some of the tested transgenic plants. Fig. 5 shows the immunostrip result for a crude protein extracted from control and transgenic plants. Table 2 shows a summary of the immunostrip detection of Cry1Ac protein for all tested transgenic plants.

Table 2.

Summary of Immunostrip detection of Cry1Ac protein in the progenies of Cry1Ac pea plants (Continuation)

      Immunostrip signal
       Immunostrip signal
No. Transgenic line Progeny level Control Test No. Transgenic line Progeny level Control Test
37 C5-2-2-1 T3 + + 54 DqR-8-4-3 T3 + +
38 D2R-2-12-1 T3 + + 55 DqR-8-4-4 T3 + +
39 DA-2-1-1 T3 + + 56 DqR-8-4-5 T3 + +
40 DA-2-1-1-1 T4 + + 57 DqR-8-4-6 T3 + −
41 DA-2-1-2 T3 + + 58 DqR-8-6 T2 + −
42 DA-2-1-3 T3 + − 59 DqR-8-7 T2 + +
43 DA-2-1-4 T3 + + 60 DqR-8-7-1 T3 + +
44 DA-2-1-4-1 T4 + + 61 DqR-8-7-2 T3 + +
45 DA-2-1-5 T3 + − 62 DqR-8-7-2-1 T4 + +
46 DA-2-1-6 T3 + − 63 DR21R-2-2 T3 + −
47 DA-2-2 T2 + − 64 E1 5(a)-2-1 T2 + +
48 DA-2-3-6 T3 + + 65 G/A-1-1 T2 + +
49 DA-2-3-6-1 T4 + + 66 G51-1-11-1 T3 + +
50 DA-2-3-9 T3 + + 67 G51-2-2-3 T3 + +
51 DA-2-3-9-1 T4 + + 68 G51-2-2-3-1 T4 + +
52 DqR-8-4-1 T3 + + 69 G51-2-2-3-1-1 T5 + +
53 DqR-8-4-2 T3 + +          
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Figure 5.

Figure 5.

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Immunostrip detection of Cry1Ac protein in control and transgenic pea plants.

Table 2.

Summary of Immunostrip detection of Cry1Ac protein in the progenies of Cry1Ac pea plants.

      Immunostrip signal
       Immunostrip signal
No. Transgenic line Progeny level Control Test No. Transgenic line Progeny level Control Test
1 Control − + − 36 C1-2-6-15-1 T4 + +
2 A2/D12-1-1 T2 + + 37 C5-2-2-1 T3 + +
3 A2/D12-1-1-1 T3 + + 38 D2R-2-12-1 T3 + +
4 A2/D12-1-1-2 T3 + + 39 DA-2-1-1 T3 + +
5 A2/D12-1-1-1-1 T4 + + 40 DA-2-1-1-1 T4 + +
6 B1-2-1 T2 + − 41 DA-2-1-2 T3 + +
7 B3-1-4-2 T3 + − 42 DA-2-1-3 T3 + −
8 B3-1-4-3 T3 + − 43 DA-2-1-4 T3 + +
9 B3-1-4-4 T3 + − 44 DA-2-1-4-1 T4 + +
10 B3-1-4-5 T3 + + 45 DA-2-1-5 T3 + −
11 B3-1-4-6 T3 + + 46 DA-2-1-6 T3 + −
12 B3-1-11-4-1 T4 + − 47 DA-2-2 T2 + −
13 B3-3-1-1 T3 + − 48 DA-2-3-6 T3 + +
14 B3-3-1-2 T3 + − 49 DA-2-3-6-1 T4 + +
15 B3-3-1-3 T3 + − 50 DA-2-3-9 T3 + +
16 B3-3-1-4 T3 + − 51 DA-2-3-9-1 T4 + +
17 B3-3-1-5 T3 + − 52 DqR-8-4-1 T3 + +
18 B3-3-2-1 T3 + − 53 DqR-8-4-2 T3 + +
19 B3-3-2-4 T3 + − 54 DqR-8-4-3 T3 + +
20 BR-5-1 T2 + + 55 DqR-8-4-4 T3 + +
21 BR-5-1-1 T3 + + 56 DqR-8-4-5 T3 + +
22 BR-5-1-4 T3 + + 57 DqR-8-4-6 T3 + −
23 BR-5-1-4-1 T4 + + 58 DqR-8-6 T2 + −
24 C1-1-2 T2 + − 59 DqR-8-7 T2 + +
25 C1-2-1-6-13-1-1 T6 + − 60 DqR-8-7-1 T3 + +
26 C1-2-1-6-13-1-2 T6 + + 61 DqR-8-7-2 T3 + +
27 C1-2-1-6-13-1-3-1 T7 + + 62 DqR-8-7-2-1 T4 + +
28 C1-2-1-6-13-1-3-6 T7 + + 63 DR21R-2-2 T3 + −
29 C1-2-1-6-13-1-3-6-1 T8 + + 64 E1 5(a)-2-1 T2 + +
30 C1-2-3-3-3 T4 + + 65 G/A-1-1 T2 + +
31 C1-2-3-3-3-1 T5 + + 66 G51-1-11-1 T3 + +
32 C1-2-3-3-3-2 T5 + + 67 G51-2-2-3 T3 + +
33 C1-2-3-3-3-2-1 T6 + + 68 G51-2-2-3-1 T4 + +
34 C1-2-3-3-3-3 T5 + + 69 G51-2-2-3-1-1 T5 + +
35 C1-2-3-6-2 T4 + +          
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Leaf Paint Functional Characterization of Cry1Ac Transgenic Pea Lines

Figure 6 shows the result of the leaf paint assay for some of the plants. In the progenies of transgenic pea lines, both herbicide tolerant (complete or partial) and susceptible plants were observed (Fig. 6a-h). The leaves of all control plants were susceptible to the applied herbicide solution (Fig. 6i). The result demonstrated the functionality of the bar gene product in the developed transgenic lines.

Figure 6.

Figure 6.

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Leaf paint functional assay result for some of the assayed plants showing herbicide tolerant (a-e: complete and f: partial) and susceptible (g-h) transgenic plants from different transgenic lines and herbicide susceptible non-transgenic control plant (i). The red arrows indicate the herbicide solution treated leaves.

Evaluation of Insect Resistance of the Cry1Ac Transgenic Pea Lines

Larval Mortality on Cry1Ac Transgenic Pea Plants

Progenies from different transgenic lines were subjected to insect bioassays using larvae of Heliothis virescens. Table 3 shows the result of larval mortality studies on the different transgenic lines. Total larval mortality was recorded on 5 of the transgenic lines. Of the tested lines, 4 lines showed about 73-92% larval mortality, while 3 lines showed less than 20% larval mortality. On one transgenic line (C7), the recorded mortality rate was less than that observed on control plants (17.42%).

Table 3.

Larval mortality on different transgenic pea lines.

      Number of larvae
    
Plant line Progeny level used Number of plants+ Inoculated Survived Died Mortality rate (%) Corrected mortality rate (%)a
Control plants   31 155 128 27 17.42  
Transgenic lines              
A2/D12-1 T2-T4 4 20 2 18 90 87.89
A2R-2-1 T2 1 5 4 1 20 3.13
B1 T2 2 10 7 3 30 15.23
B20R-2-1 T2 1 5 2 3 60 51.56
B3 T3-T4 14 70 38 32 45.71 34.26
BR-5 T4 2 10 0 10 100 100.00
C1 T3-T7 10 50 11 39 78 73.36
C5 T2-T4 4 20 15 5 25 9.18
C7 T2-T3 2 10 9 1 10 −8.98*
C8R T2 1 5 2 3 60 51.56
D2R T2-T3 2 10 0 10 100 100.00
DA T2-T4 10 50 6 44 88 85.47
DqR T2-T4 6 30 2 28 93.33 91.93
E15 T2 1 5 0 5 100 100.00
G/A T2 1 5 0 5 100 100.00
G51 T3-T5 7 35 0 35 100 100.00
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+

Transgenic progenies from the same line were pooled together.

a

Corrected mortality was calculated using the following formula as described in Zhang & Tang 2004: Corrected Mortality rate on transgenic plants (%) = [(LMRT-LMRC)/(100-MRC)] × 100; Where LMRT: Larva Mortality rate (%) on transgenic plant, LMRC: Larva mortality rate (%) on control plants.

*

line C7 had problem in estimating feeding damage.

Larval Feeding Damage on Cry1Ac Transgenic Pea Plants

Similar to larva survival, the average feeding damage on transgenic plants varied from line to line (Table 4). Fig. 7 shows relative feeding damage on the control and transgenic plants. On the transgenic plants, the average feeding damage ranged from very negligible (recorded as 5%) to 57.50%. On plants from 9 transgenic lines, the estimated feeding damage was about 5-11%. More than 50% average feeding damage was recorded on plants from 2 transgenic lines. Among the transgenic plants, the maximum feeding damage was observed on the transgenic plants from the C7-line. On control plants, the average feeding damage caused by the inoculated larvae was about 53.06%. More than 70% (22 out of 31) of the control plants incurred 50% or more feeding damage. In the transgenic lines, 9 plants from 4 transgenic lines incurred 50% or more feeding damage.

Table 4.

Estimated feeding damage on different transgenic lines.

      Number of plants with estimated feeding damage of
  
Plant line Progeny level used Number of plants+ 0-10% 11-20% 21-49% 50-100 % Average estimated feeding damage (%)
Control plants   31 1 1 7 22 53.06
Transgenic lines              
A2/D12-1 T2-T4 4 4 0 0 0 5.00
A2R-2-1 T2 1 0 0 1 0 35.00
B1-line T2 2 0 0 1 1 55.00
B20R-2-1 T2 1 0 0 1 0 25.00
B3 T3-T4 14 2 6 1 5 28.93
BR-5 T4 2 2 0 0 0 5.00
C1 T3-T7 10 6 3 1 0 11.50
C5 T2-T4 4 0 1 1 2 46.25
C7 T2-T3 2 0 1 0 1 57.50
D2R T2-T3 2 2 0 0 0 5.00
DA T2-T4 10 10 0 0 0 5.50
DqR T2-T4 6 6 0 0 0 5.00
E15 T2 1 1 0 0 0 5.00
G/A T2 1 1 0 0 0 5.00
G51 T3-T5 7 7 0 0 0 5.00
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+

Transgenic progenies from the same line were pooled together.

Figure 7.

Figure 7.

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Larval feeding damage on non-transgenic control plant and cry1Ac transgenic plants after one week of larvae feeding. The number in the bracket indicated the estimated plant damage due to the inoculated larvae feeding.

Discussion

Pea has been manipulated in vitro since Schroeder et al. Schroeder et al., 1993 have reported regeneration and transformation protocol. Today, pea is one of the crop plants which have a routine regeneration and transformation protocol based on Agrobacterium-mediated transformation. The cry1Ac transgenic pea lines Negawo 2012 used in this study are also the result from the application of such established protocol.

Inheritance and expression of cry1Ac gene: Progenies of different cry1Ac transgenic pea lines have been characterized using molecular and functional analyses. The results demonstrated the integration, inheritance, expression as well as the functionality of the introduced T-DNA region (cry1Ac and bar genes). The use of transgenic plants depends on the faithful inheritance and expression of the novel traits. Finnegan, Mcelroy 1994; Meyer 1995 Despite the genomic integration, the expression of a transgene could vary from line to line and/or could be silenced completely. Meyer 1995; Kohli, Miro, Twyman 2010; Kohli 2006 As reviewed by different authors, Kohli, Miro, Twyman 2010; Kohli 2006; Iyer, Kumpatla, Chandrasekharan, Hall 2000 variation in transgene expression has been reported in many transgenic plants such as petunia, tobacco, rice, wheat and maize. Similarly, variation in the expression of cry1Ac gene was observed among the different transgenic lines. In the progeny of some transgenic lines (such as C7-1-3-1, C7-1-4-1, C5-1-1 and C5-3-1), despite that the integration and inheritance of the cry1Ac gene was demonstrated by PCR, no expression of cry1Ac gene was observed at the transcriptional level (RT-PCR result). This is also supported by the negative result of the immunostrip assays and/or leaf paint assay for the bar gene product. Possibly the T-DNA region has been silenced in these transgenic plants. Transgene silencing is a common phenomenon which can be due to copy number, integration pattern, integration region and/or epigenetic effects and can occur at the transcriptional and/or post-transcriptional level. Finnegan, Mcelroy 1994; Kohli, Miro, Twyman 2010, Stam et al., 1997 For example, in the transgenic line B3 and its progenies, Southern blot analysis showed the presence of multiple copies of the transgene. Negawo 2012 Most of the progenies from this line, however, showed negative results for the immunostrip and LP assays. Hence, without ruling out other factors, it might be possible that the copy number contributed to the silencing of the T-DNA in these plants. Transgene integration is a random process in which the transgene is inserted anywhere in the host genome Kohli 2006; Wallroth, Gerats, Rogers, Fraley, Horsch 1986, Chyi et al., 1986 with consequences on the expression levels. Kohli, Miro, Twyman 2010; Kohli 2006, Stam et al., 1997 In petunia, the integration of transgene in the highly repetitive DNA and methylated region of the genome has caused inactivation of the integrated transgene. Prols, Meyer 1992

Insect Resistance Evaluation of the Cry1Ac Transgenic Pea Lines

In support of the molecular and leaf paint functional analyses, the insect bioassay results clearly demonstrated the functionality of the Cry1Ac protein in some of the analyzed transgenic lines. Up to 100% larval mortality as well as substantially reduced plant damage was observed on the transgenic plants expressing the transgene indicating the insect resistance of the developed transgenic lines. On the other hand, on transgenic plants in which the T-DNA region seemed to be silenced (negative RT-PCR and LP results) or expressed at very low levels (qRT-PCR results), the insect bioassay showed the susceptibility of the plants to the larval challenge. For example, in transgenic plants A2R-2-1 and B3-3-1-5 the result of qRT-PCR showed very low expression of cry1Ac gene. Similarly, transgenic plants C7-1-4-1, C7-1-4-2, B1-1-1, B3-3-1-5 and A2R-2-1 showed susceptibility to the applied herbicide solution. On these plants, less larval mortality rate (1-2 out of 5 larvae) and relatively high feeding damages (29-57.5 %) were recorded.

Similar results have been reported on other transgenic crops expressing cry1Ac or other cry genes. Reduced feeding rates to complete larval mortality of Manduca sexta was reported on transgenic tobacco plants expressing Bt crystal proteins. Barton, Whiteley, Yang 1987; Vaeck et al, 1987 More recently, complete mortality of larvae of 2 insect pests (Manduca sexta and Heliothis virescens) and negligible leaf damage was observed on transgenic tobacco plants expressing cry1Ac gene under the control of wound-inducible promoter. Gulbitti-Onarici et al., 2009 Up to 100% larval mortality of Lepidopteran insect pests (Manduca sexta, Heliothis virescens, Helicoverpa armigera and Spodoptera litura) and very little sign of feeding damage has been reported on transgenic tomato plants expressing crystal toxin from Bt var. Kurstaki HD-1 Fischhoff et al., 1987 and Cry1Ab protein. Koul et al., 2014 Total larval mortality of 2 insect pests (Trichoplusia ni and Spodoptera exigua ) and high levels of plant protection have been observed on transgenic cotton plants expressing cry1Ac or cry1Ab genes from Bt var Kurstaki (HD-1 and HG-73, respectively). Perlak et al., 1990 Different cry toxins (cry1Ab, cry1ac or cry1B) have also been expressed in cereal crops like rice Cheng, Sardana, Kaplan, Altosaar 1998; Datta et al., 1998; Breitler 2001, Khanna, Raina 2002; Ramesh, Nagadhara, Reddy, Rao 2004, maize Bohorova et al., 1999; Armstrong 1995, Koziel et al., 1993 and sorghum.Girijashankar et al., 2005 According to the reports, up to 100% larval mortality of the target insect pests as well as substantially reduced plant damage was observed on the transgenic plants of these crops.

Conclusion and Future Outlook

The scope of this study was limited to laboratory and greenhouse experiments. It demonstrated the successful development of insect resistant transgenic pea lines expressing insecticidal Cry1Ac protein from soil bacterium Bacillus thuringiensis. Field trials were not conducted on the developed transgenic lines. Hence, further research activities are required to evaluate the field performance of the transgenic lines under natural growing conditions. Then, selected transgenic lines could be used in production programs to reduce yield losses due to the target pest or in improvement programs to combine with other traits for multiple resistances.

Materials and Methods

Plant Material

Seeds of cry1Ac transgenic pea (Pisum sativum L. cv. sponsor) lines Negawo 2012 were used for molecular and functional characterizations. The transgenic lines were developed using the routine pea transformation protocol using Agrobacterium tumefaciens strain EHA105 harboring a pSoup pGII35SCry1Ac vector Schroeder et al., 1993 with modification as described in Richter et al. Richter et al., 2006 The vector contains bar gene as selectable marker (antibiotic selectable markers were avoided according to European Directive 2001/83/EC), which encodes for the enzyme phosphinothricin acetyltransferase (PAT) that inactivate the total herbicide Basta®. Thompson et al., 1987

Molecular Analyses of Transgene Presence and Expression

Cry1ac transgenic pea lines and their progenies were characterized using PCR. Genomic DNA was isolated from fresh leaves using quick method Edwards et al., 1991 as described in Kumari et al. Kumari et al., 2012 Primers used for PCR amplification are indicated in Negawo et al. Cheng et al., 1998 They are Cry-750-For: 5′-GTTCAGGAGAGAATTGACCC-3′ and Cry750-Rev: 5′-CTTCACTGCAGGGATTTGAG-3′ amplifying 750 bp of cry1Ac gene and HMG-For: 5′-ATGGCAACAAGAGAGGTTAA-3′and HMG-Rev: 5′-TGGTGCATTAGGATCCTTAG-3′ amplifying 570/350 bp of pea housekeeping HMG-I/Y gene (DNA/cDNA), which was used as reference. Gupta et al., 1997 RNA was isolated from young leaves using NucleoSpin® RNA plant kit (Macherey-Nagel) and used for cDNA synthesis using RevertAid™ H Minus First Strand cDNA Synthesis kit (Thermo Scientific). The synthesized cDNA was used for detection of cry1Ac gene expression using RT-PCR and qRT-PCR.

Primers (Cry160-For: 5′-GATTGGAAACTACACCGACC-3′ and Cry160-Rev: 5′-GGAGTCATAGTTCGGGAAGA −3′) amplifying 160 bp of the cry1Ac gene sequence were designed and used for the quantitative analysis of cry1Ac transgene expression in the transgenic plants. To normalize the expression of cry1Ac transgene, pea housekeeping gene (HMG-I/Y) Gupta et al., 1997 were used as reference using the following primers HMGIII-For: 5‘-AGGGGTAGGCCGAAGAAGAT-3′ and HMGIII-Rev: 5′-TGAGGCTTCACCTTAGGAGG −3′ amplifying 164 bp.

The qRT-PCR was performed on iQ™ 96-well PCR plates covered with Optical Sealing Tape (Bio-Rad) on iCycler iQ5 Real Time PCR detection system (Bio-Rad). The qRT-PCR mixture contained 3 pMol (0.3 µl from 10 pMol stock) of both forward and reverse primers, 7.5 µl of iQTM SYBR® Green Supermix (Bio-Rad) and 1 µl cDNA of each sample. The reaction volume was brought to 15 µl with sterile ddH2O.

The qRT-PCR profile included initial denaturation step of 95°C for 10 min followed by 40 cycles of 95°C for 20 sec denaturation step, 59°C for 30 sec annealing step and 72°C for 45 sec extension step and a finally melting curve analysis step of 55-95°C for 10 sec with 0.5 °C increment after each temperature point.

During the qRT-PCR analysis, each cDNA sample was replicated 3 times. The qRT-PCR data were automatically collected and analyzed using iQ5 Optical System Software v2.0 (Bio-Rad). The unscaled normalized expression relative to the control plant was chosen in the gene expression analysis option.

Detection of Cry1Ac Protein in Transgenic Plants

Qualitative detection of Cry1Ac protein in the leaves of transgenic plants was conducted using a commercial immunostrip specific to Cry1Ab/Ac protein (Bt-Cry1Ab/1Ac Immunostrip® test, Agdia Inc.) according to the manufacturer's instruction.

Insect Bioassay

For insect bioassay experiments, larvae of Heliothis virescens (kindly provided by Dr. Jürgen Langewald, BASF Plant Science, Limburgerhof, Germany) were used to test the resistance and/or susceptibility of the transgenic lines. Heliothis virescens is a polyphagous insect pest which can feed and incur damage to many plant species including legumes.

Seeds of both transgenic pea lines and control none-transgenic plants were grown in growth chamber (22±2 °C, 16/8 photoperiod). Before larvae inoculation, the transgenic nature of the plants was confirmed as described above. Leaf paint assay was used to characterize the transgenic plants to assess the functionality of the bar gene product as described in Richter et al. Richter et al., 2006 Then, 5 larvae of Heliothis virescens were inoculated on each of the selected pea plants. The inoculated plants were covered with a glass cylinder to restrict the larvae movement (Fig. 8). Then, larval mortality and feeding damage were recorded on each plant one week after larvae inoculation.

Figure 8.

Figure 8.

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Larvae inoculated plants covered with a glass cylinder to restrict larvae movement.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

AcknowledgmentS

We would like to thank Dr. Jürgen Langewald (BASF Plant Science, Limburgerhof, Germany) for kindly providing the larvae of tobacco budworm (Heliothis virescens). We also like to thank Mr. Timo Michel for his assistance on rearing the insect and subsequent feeding experiments.

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