EVALUATION OF TRANSGENIC POTATO LINES RESISTANCE TO POTATO TUBER MOTH, Phthorimaea operculella (ZELLER)

he potato, Solanum tuberosum L., is one of the world’s most important food crops, following rice, wheat, and maize (Ross, 1986). In Egypt, the production reaches up to 3 million metric tons annually. Potato is also the leading vegetable export in Egypt, with '225,000 metric tons exported to the European market. The potato tuber moth, Phthorimaea operculella (Zeller), (PTM) is the most serious insect pest of potatoes worldwide. It is probably originated in South America, where the potato also originates (Goldson and Emberson, 1985). In the field, the moths lay their eggs on the potato foliage and the larvae mine the foliage and the stems. This feeding damage extends to storage, reducing potato quality and increasing the potential for pathogen infection. In India, tuber damage in storage ranges from 25 to 100% (Saxena and Rizvi, 1974) and tubers are 100% damaged in 1.5 to 2 months in Tunisia (Essamet et al., 1988) while in Egypt, the potato losses reach up to 100% in fields as well as in storage (Ahmed et al., 2013). In Sudan, 30-40% of the potatoes are stored in underground pits which could be completely destroyed by tuber moth within 2 months (Ali, 1993). The Bacillus thuringiensis Crystal proteins are non-toxic to humans, animals, birds, and most beneficial insects and have been used as biological insecticides to control agricultural insect pests (Koziel et al., 1993). However, field applications of this biological pesticide have not always provided a cost-effective control of insect pests. The genes (cry genes) which encode Bt crystal proteins have been cloned, sequenced, and transformed into various crops and are now available commercially (Barton and Miller, 1993). Jansens et al. (1995) was the first to develop Bttransgenic potato lines resistant to PTM using both native and modified truncated cryIA(b) gene. Further potato transgenic lines expressing truncated cryIA(b) gene was developed and evaluated by Canedo et al. (1999). Field trials of transgenic lines were carried out in two Latin America countries; San Ramón (Bolivia) in 1995 and Tacna (Peru) in 1997-1998. In both trials, transgenic potato lines showed a high resistance level against PTM attack.

he potato, Solanum tuberosum L., is one of the world's most important food crops, following rice, wheat, and maize (Ross, 1986). In Egypt, the production reaches up to 3 million metric tons annually. Potato is also the leading vegetable export in Egypt, with '225,000 metric tons exported to the European market. The potato tuber moth, Phthorimaea operculella (Zeller), (PTM) is the most serious insect pest of potatoes worldwide. It is probably originated in South America, where the potato also originates (Goldson and Emberson, 1985). In the field, the moths lay their eggs on the potato foliage and the larvae mine the foliage and the stems. This feeding damage extends to storage, reducing potato quality and increasing the potential for pathogen infection. In India, tuber damage in storage ranges from 25 to 100% (Saxena and Rizvi, 1974) and tubers are 100% damaged in 1.5 to 2 months in Tunisia (Essamet et al., 1988) while in Egypt, the potato losses reach up to 100% in fields as well as in storage (Ahmed et al., 2013). In Sudan, 30-40% of the potatoes are stored in underground pits which could be completely destroyed by tuber moth within 2 months (Ali, 1993).
The Bacillus thuringiensis Crystal proteins are non-toxic to humans, animals, birds, and most beneficial insects and have been used as biological insecticides to control agricultural insect pests (Koziel et al., 1993). However, field applications of this biological pesticide have not always provided a cost-effective control of insect pests. The genes (cry genes) which encode Bt crystal proteins have been cloned, sequenced, and transformed into various crops and are now available commercially (Barton and Miller, 1993). Jansens et al. (1995) was the first to develop Bttransgenic potato lines resistant to PTM using both native and modified truncated cryIA(b) gene. Further potato transgenic lines expressing truncated cryIA(b) gene was developed and evaluated by Canedo et al. (1999). Field trials of transgenic lines were carried out in two Latin America countries; San Ramón (Bolivia) in 1995 and Tacna (Peru) in 1997-1998. In both trials, transgenic potato lines showed a high resistance level against PTM attack.
Three potato cultivars, 'Lemhi Russet', 'Atlantic' and 'Spunta', were transformed with five different vectors encoding Bt-cry5 gene under different T promoters (Douches et al., 1998;Mohammed et al., 2000). The 'Spunta' transgenic tubers expressing the Bt-cry5 under the patatin element were not successful whereas the mortality range was 25.6 and 31.1%. Two out of three Spunta lines transformed with 35S/Bt-cry5 gene construct (G2 and G3) showed 100% mortality and other two lines with the constitutive Gelvin super promoter (S1 and S4) had the same mortality level. Furthermore, field trials on Bt-cry5 transgenic potato lines were carried out in three different locations; one in the USA (Montcalm Research Farm at Michigan) and two in Egypt (AGERI at Giza and CIP at Kafr El-zayat), between 1997 and 2001 (Douches et al., 2004). None of the five Bt-cry5-Atlantic lines were significantly different in potato PTM damage from 'Spunta', whereas most of the Bt-cry5-Spunta lines were different. 'Spunta G2', 'Spunta G3', 'Spunta-S1', 'Spunta-S4' and 'Spunta 6a-3' were free of PTM infestation. The 'Spunta G2' was selected for commercialization, therefore, field trials were performed in seven locations in South Africa over five years (Douches et al., 2010;Visser et al., 2013) and food safety and biosafety issues were also discussed (Douches et al., 2008).
The food sand feed safety for 'Spunta G2', 'Spunta G3' and 'Spunta 6a-3' were previously studied by Metry et al. (2008). The amino acids profile as well as vitamins and minerals contents of the three transgenic lines don't significantly different from Spunta. The 'Spunta G2'is extensively studied for commercialization by Douches' group, therefore, it is excluded from the field study. Herein, both 'Spunta G3' and 'Spunta 6a-3' are studied for their resistance to PTM infestation and agronomical performance in the field before proceeding towards registration for local commercial release.
Adaptation and acclimatization of transgenic potato lines and control were carried out in the Bio-containment greenhouse facilities at AGERI according to Metry et al. (2003).

In vitro tuberization
The micropropagated plantlets were transferred to first liquid medium containing MS salt components supplemented with gibberellic acid (GA 3 ), 20 g/l sucrose, 500 μl benzyl adenine (BA), 10 μl naphthalene acetic acid (NAA). The plantlets were then transferred into second liquid medium consisted of MS medium supplemented with 80 g/1 sucrose, 5 ml benzyl adenine (BA), 1 mg/l, chlorocholine chloride (CCC) for 50 days in complete dark (Huiqun, 2009). The medium encourage and support the in vitro micro tuber formation. The microtubers were harvested and sorted out into small and large sizes and the length and width of tubers were determined and analyzed used standard error method.

Molecular analysis
PCR: Genomic DNA was extracted from the leaves of three transgenic potato lines ('Spunta G2', 'Spunta G3' and 'Spunta 6a-3') as well as nontransgenic 'Spunta' using DNAeasy plant extraction kit (Qiagen, USA) as described in the manufacturer's instructions provided with kit. A fragment of 732bp cry5 gene was amplified using the gDNA extracted from the three transgenic lines and specific primer; Cry5s-476F; 5'-GCAGCGTGGTGAAGAGCA-3' and Cry5s-1208R; 5'-ACACGTCGCG GCTGG TGAAG-3'. The reaction conditions included 40 cycles of, denaturation at 94C for 1 min, annealing at 58C for 1 min and extension at 72C for 1.5 min. An aliquot of 10 l PCR product were analyzed on 1% agarose gel.

RT-PCR:
Total RNA was extracted from the leaves of the three lines as well as non-transgenic using SV total RNA isolation system (Promega, USA) as described in the manufacturer's instructions provided with kit. The total RNA from each line was reverse transcribed using a Su-perScript ™ II Reverse transcriptase (Invitrogen, USA) according to the manufacturer's instructions. The RNA template was removed from the 1 st stand cDNA by 2 units of E. coli RNaseH (Promega) for 20 min at 37C. The 1 st strand cDNA was mixed with 2.5 units of Taq polymerase (Promega) in 1x reaction buffer, 0.5 M dNTPs, 25 mM MgCl 2 and 25 pmoles primers. The amplification conditions were 94C for 2 min followed by 40 cycles of 94C for 15 sec, 60C for 15 sec and 72C for 30 sec followed by 4 min at 72C. The RT-PCR amplification was performed for using cry5-specific primer set mentioned is the previous section.

AFLP:
Genomic DNA was prepared from the leaflets of each transgenic and control line. DNA extraction, purification and bulking were performed as described by Hussein et al. (2005).
The AFLP procedure was performed, with minor modifications, accord-ing to the protocol supplied with the AFLP Analysis System I (Invitrogen, USA) manual. Approximately 400 ηg of genomic DNA of each of the four samples was digested with EcoRI and MseI at 37C for 2 hours. EcoRI and MseI adaptors were ligated to the digested DNA samples to generate template DNA for amplification. Pre-amplification was carried out using 5 µl of diluted ligation products with 5-primer combinations each carrying one selective nucleotide with the following conditions, 20 cycles of 94C for 30 sec, 56C for 60 sec and 72C for 60 sec. Five microliters of 50 times diluted PCR products was used for selective AFLP amplification using EcoRI and MseI adaptor-specific primer set, each carrying three selective nucleotides. The PCR selective amplification condition was as follows: one cycle of 94C for 30 sec, 65C for 30 sec, and 72C for 60 sec followed by 12 cycles of touchdown PCR in which the annealing temperature was decreased by 0.7C every single cycle until annealing temperature goes down to 56C.
Once the annealing temperature at 56C, another 23 cycles of pre-amplification conditions were applied to the reaction.
Two l of the reaction product was mixed with an equal volume of formamide loading buffer (98% [v/v] formamide, 10 mM EDTA, 0.005% [v/v] of each of xylene cyanol and bromophenol blue), denatured by incubating at 90C for 5 min and quickly cooled on ice. The products were analyzed on 6% (w/v) denaturing polyacrylamide gels. The gel was run at constant power (50-55 W) until the xylene cyanol was about two-thirds down the length of the gel. The gel was silver stained using Silver Sequence DNA Staining Reagents (Promega, USA) according to the protocol described by the manufacturer.

Field trials
Two transgenic lines ('Spunta G3' and 'Spunta 6a-3') along with nontransgenic potatoes were field tested to evaluate resistance to potato tuber moth and assess their morphological characteristics. Field Trial was carried out between November, 2011 and April, 2012 at AGERI farm, Giza, Egypt.
The experiment was three plot replicates, each with seven rows of 15 plants. Each row is 3 m long, 0.5 m space among adjacent rows, and 0.2 m plant-spacing. The transgenic lines as well as the control line were randomly distributed among the replicates; however, the tested line was planted in ranked spot only once. Plant growth was evaluated by plant height and branches and yield data was also recorded.
The field experiment was artificially challenged with PTM by seeding the plots with infested tubers. Potato tubers were infested at the lab, 25 neonate larvae were added to each tuber and allowed to grow at the room temperature. After 12 days, the infested tubers were collected and disturbed in the field. Three infested tubers were added to each row in the vicinity of plants. The experiment was PTM-challenged twice, the first challenge during the first week of March and repeat-ed four weeks later. At harvest, tubers were evaluated for PTM damage by checking infestation symptoms of the tubers.

In vitro micropropagation
The transgenic lines; 'Spunta G2', 'Spunta G3' and 'Spunta 6a-3' as well as non-transgenic 'Spunta' were successfully micropropagated in vitro using nodal cuttings technique. The method utilize MS basic salt medium supplemented with gibberellic acid (GA 3 ). Nodal cuttings were sub-cultured repeatedly on a fresh medium after 3 weeks. The appearance of in vitro plantlets was normal.

In vitro tuberization
The microtubers were harvested and sorted out according to size (Fig. 1). Both length and width of each size were measured and recorded (Table 1). The data was analyzed using standard error method. Results revealed that there is no significant difference of the microtuber dimensions among the three transgenic lines compared to non-transgenic potatoes. Both length and width of large and small sizes are represented in Fig. (2). The length of 'Spunta 6a-3' microtubers is smallest among the lines of both small and large size of 0.64±0.17 and 1.21± 0.26, respectively. The 'Spunta G3' shows slightly tallest small size microtubers of 0.71±0.14 while tallest large size microtubers belong to control 1.38±0.35. Other dimension parameter which is the microtuber's width showed almost the same dimension for the small size 'Spunta' control and 'Spunta G3'. Morphological characteristics of the microtubers are within the normal range of variability compared to control plants.

PCR and RT-PCR
Both PCR and RT-PCR analysis were performed on gDNA and mRNA, respectively, prepared from transgenic lines as well as 'Spunta' cultivar. All PCR and RT-PCR reactions of cry5-transgenic lines resulted in positive amplifications of 732 bp using cry5-specific primers while non-transgenic showed no PCR product (Fig. 3A & B). These results indicate the presence of active transcripts of cry5 gene within tissues of 'Spunta G2', 'Spunta G3' and 'Spunta 6a-3' and its lack within 'Spunta'. PCR analysis was previously used to detect both cry1A(b) and nptII genes within transgenic potatoes against PTM (Hagh et al., 2009) while southern bolt was selected to detect the same gene in different transgenic potatoes (Canedo et al., 1999). PCR analysis was also used in segregation and efficacy studies for cry gene in 'SpuntaG2' .

AFLP
The banding patterns generated by AFLP analyses were examined to determine the level of polymorphism and the genetic relatedness between control and the three transgenic plants using five selective primer combinations. The amplified fragments were scored as present (1) or absent (0). The size of AFLP fragments generated by the different primer combinations ranged from 50-500 bp (Fig. 4). A total number of 234 bands were generated from 5 AFLP primer combinations (Table   2)  is more than 97% (Chandel et al., 2010).
They also concluded that "the transgenic plants developed by Agrobacterium tumefaciens infection than that of developed through particle bombardment".
Similar results were also revealed by AFLP that showed limited genomic changes among three transgenic sugarcane populations produced by Agrobacterium infection and non transgenic sugarcane (Carmona et al., 2005).

Field trials
AGERI is located within populated area and away from cultivated lands; therefore the agricultural insect pest population is too low to cause noticeable damage. To overcome lack of natural infestation of PTM, the cry5-transgenic potato lines and the control were artificially infested twice with PTM-infected tubers in the field. Assessments of field challenge of both 'Spunta G3' and 'Spunta 6a-3' showed high resistance level against PTM attack, only 3% of the tuber yield is susceptible to larval penetration (Table 3). Results from field data showed the similar trends as those from laboratory assay, field trials and during storage experiments. Previous results showed that resistance of the transgenic lines to PTM infection reach to 100% (Mohammed et al., 2000, Douches et al., 2004Randon et al., 2009;Douches et al., 2010).
The high mortality level of these transgenic lines is due to high expression level of the codon modified Bt-cry5 that is regulated by the constitutive CaM35S promoter. Constitutive promoters have been deployed to drive cry gene expression in transgenic potatoes resistant to PTM (Canedo et al., 1999;Douches et al., 1998Douches et al., , 2002Douches et al., and 2013Mohammed et al., 2000). However, other inducible promoters such as light-inducible promoter, Lhca3, maize light-inducible promoter, PEPC, (Meiyalaghan et al., 2006) and Green-tissue-specific, C4-PEPC-promoter (Hagh et al., 2009) were also used to regulate cry genes for resistance to the same pest.
Because the field trials were based on planting microtubers, the yield for both transgenic and control is low compared to regular size tubers. The phenotypic variations with respect to plant height and leaf length/weight (L:W) ratio was hard to be determined. The agronomic performance is based on tubers yield of the control and the two transgenic lines. The average of tubers yield of 'Spunta G3' and 'Spunta 6a-3' showed no significant differences compared to that of 'Spunta' (Table 4). Previous agronomic trials in Michigan and Egypt showed that many of the Bt-cry5 transgenic lines perform similarly to their non-transformed cultivar (Douches et al., 2004) and the same trend of results was observed for 'Spunta G2' (Douches et al., 2010). However, large scale agronomic trials and more parameters such as total yield/feddan, tuber size category, and specific gravity should be considered in future field trials.
The genetically enhanced potato lines undergo the mandatory pathway determined by the National Biosafety Committee which includes environmental risk assessment and food safety. This type of assessment includes; molecular characterization to determine the inserted gene and the expressed protein, nutritional composition, toxicological and histo-pathological evaluation and allergenicity tests. Moreover, the effects of genetically modified potato lines on the microbial communities in different types of soil and its impact assessment on soil ecosystem are also considered. Molecular analysis of the inserted gene and field trials are presented in the current paper while other parameters to be evaluated are in process.

SUMMARY
The potato tuber moth, Phthorimaea operculella (Zeller), is the major destructive insect of potato. The larvae attack foliage and tubers in the field and in storage as well. Over the past years, there was a tremendous effort to produce transgenic potato cultivar that is resistant to the larval infestation of potato tuber moth. 'Spunta' potato cultivar was previously transformed with Bt-cry5 gene under 35S promoter construct. Three transgenic 'Spunta' lines, 'Spunta G2', 'Spunta G3' and 'Spunta 6a-3', were selected for their high resistance to PTM attack. After 14 years of storage, these three lines are subjected to in vitro micropropagation, tuberization and re-evaluation. Both PCR and RT-PCR confirmed the presence of cry5 gene within 'Spunta' genome and its expression within potato plant tissues. Amplified fragment length polymorphism (AFLP) analysis revealed that the percentage of polymorphism among the three lines and non-transgenic ranged from 0% to 10.6%. Two lines 'Spunta G3' and 'Spunta 6a-3' were selected for field agronomical performances as steps for environmental safety as well as PTM challenge. Both lines are highly resistant to PTM compared to the control, only 3% of the yield shows susceptibility to larval penetration of the tubers.